Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of kinase proteins that arerelated to the protein kinase C subfamily, recombinant DNA molecules,and protein production. The present invention specifically providesnovel peptides and proteins that effect protein phosphorylation andnucleic acid molecules encoding such peptide and protein molecules, allof which are useful in the development of human therapeutics anddiagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Protein Kinases

[0003] Kinases regulate many different cell proliferation,differentiation, and signaling processes by adding phosphate groups toproteins. Uncontrolled signaling has been implicated in a variety ofdisease conditions including inflammation, cancer, arteriosclerosis, andpsoriasis. Reversible protein phosphorylation is the main strategy forcontrolling activities of eukaryotic cells. It is estimated that morethan 1000 of the 10,000 proteins active in a typical mammalian cell arephosphorylated. The high energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

[0004] The kinases comprise the largest known protein group, asuperfamily of enzymes with widely varied functions and specificities.They are usually named after their substrate, their regulatorymolecules, or some aspect of a mutant phenotype. With regard tosubstrates, the protein kinases may be roughly divided into two groups;those that phosphorylate tyrosine residues (protein tyrosine kinases,PTK) and those that phosphorylate serine or threonine residues(serine/threonine kinases, STK). A few protein kinases have dualspecificity and phosphorylate threonine and tyrosine residues. Almostall kinases contain a similar 250-300 amino acid catalytic domain. TheN-terminal domain, which contains subdomains I-IV, generally folds intoa two-lobed structure, which binds and orients the ATP (or GTP) donormolecule. The larger C terminal lobe, which contains subdomains VI A-XI,binds the protein substrate and carries out the transfer of the gammaphosphate from ATP to the hydroxyl group of a serine, threonine, ortyrosine residue. Subdomain V spans the two lobes.

[0005] The kinases may be categorized into families by the differentamino acid sequences (generally between 5 and 100 residues) located oneither side of, or inserted into loops of, the kinase domain. Theseadded amino acid sequences allow the regulation of each kinase as itrecognizes and interacts with its target protein. The primary structureof the kinase domains is conserved and can be further subdivided into 11subdomains. Each of the 11 subdomains contains specific residues andmotifs or patterns of amino acids that are characteristic of thatsubdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) TheProtein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego,Calif.).

[0006] The second messenger dependent protein kinases primarily mediatethe effects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

[0007] Calcium-calmodulin (CaM) dependent protein kinases are alsomembers of STK family. Calmodulin is a calcium receptor that mediatesmany calcium regulated processes by binding to target proteins inresponse to the binding of calcium. The principle target protein inthese processes is CaM dependent protein kinases. CaM-kinases areinvolved in regulation of smooth muscle contraction (MLC kinase),glycogen breakdown (phosphorylase kinase), and neurotransmission (CaMkinase I and CaM kinase II). CaM kinase I phosphorylates a variety ofsubstrates including the neurotransmitter related proteins synapsin Iand II, the gene transcription regulator, CREB, and the cystic fibrosisconductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBOJournal 14:3679-86). CaM II kinase also phosphorylates synapsin atdifferent sites, and controls the synthesis of catecholamines in thebrain through phosphorylation and activation of tyrosine hydroxylase.Many of the CaM kinases are activated by phosphorylation in addition tobinding to CaM. The kinase may autophosphorylate itself, or bephosphorylated by another kinase as part of a “kinase cascade”.

[0008] Another ligand-activated protein kinase is 5′-AMP-activatedprotein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

[0009] The mitogen-activated protein kinases (MAP) are also members ofthe STK family. MAP kinases also regulate intracellular signalingpathways. They mediate signal transduction from the cell surface to thenucleus via phosphorylation cascades. Several subgroups have beenidentified, and each manifests different substrate specificities andresponds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R.A. (1993) Nature 365:781-783). MAP kinase signaling pathways are presentin mammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1).

[0010] PRK (proliferation-related kinase) is a serum/cytokine inducibleSTK that is involved in regulation of the cell cycle and cellproliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol.Chem. 271:19402-8). PRK is related to the polo (derived from humans pologene) family of STKs implicated in cell division. PRK is downregulatedin lung tumor tissue and may be a proto-oncogene whose deregulatedexpression in normal tissue leads to oncogenic transformation. AlteredMAP kinase expression is implicated in a variety of disease conditionsincluding cancer, inflammation, immune disorders, and disordersaffecting growth and development.

[0011] The cyclin-dependent protein kinases (CDKs) are another group ofSTKs that control the progression of cells through the cell cycle.Cyclins are small regulatory proteins that act by binding to andactivating CDKs that then trigger various phases of the cell cycle byphosphorylating and activating selected proteins involved in the mitoticprocess. CDKs are unique in that they require multiple inputs to becomeactivated. In addition to the binding of cyclin, CDK activation requiresthe phosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

[0012] Protein tyrosine kinases, PTKs, specifically phosphorylatetyrosine residues on their target proteins and may be divided intotransmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs.Transmembrane protein-tyrosine kinases are receptors for most growthfactors. Binding of growth factor to the receptor activates the transferof a phosphate group from ATP to selected tyrosine side chains of thereceptor and other specific proteins. Growth factors (GF) associatedwith receptor PTKs include; epidermal GF, platelet-derived GF,fibroblast GF; hepatocyte GF, insulin and insulin-like GFs, nerve GF,vascular endothelial GF, and macrophage colony stimulating factor.

[0013] Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

[0014] Many of these PTKs were first identified as the products ofmutant oncogenes in cancer cells where their activation was no longersubject to normal cellular controls. In fact, about one third of theknown oncogenes encode PTKs, and it is well known that cellulartransformation (oncogenesis) is often accompanied by increased tyrosinephosphorylation activity (Carbonneau H and Tonks NK (1992) Annu. Rev.Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be animportant strategy in controlling some types of cancer.

[0015] Protein Kinase C

[0016] Protein kinase C (PKC) proteins are members of the STK family.Protein kinase D (PKD) proteins bind phorbol esters and diacylglyceroland are closely related to PKCs (Valverde et al., Proc Natl Acad Sci USA1994 Aug. 30;91(18):8572-6).

[0017] Protein kinase C plays a key role in modulating cellularresponses in a wide variety of extracellular receptor-mediated signaltransduction pathways, and in regulating cellular differentiation andproliferation in a wide variety of cells.

[0018] Protein kinase C genes/proteins may play an important role inmany cancers, and therefore may be useful for drug development and forscreening for, diagnosing, preventing, and/or treating a variety ofcancers. For example, tumor-specific deletions have been identifiedwithin the gene for alpha-type protein kinase C in a melanoma cell line(Linnenbach et al., Proc Natl Acad Sci USA January 1988; 85(1):74-8).Elevated expression levels of PKCs have been observed in certain tumorcell lines and it has been suggested that PKCs play an important role insignal transduction pathways related to growth control (Johannes et al.,J Biol Chem Feb. 25, 1994; 269(8):6140-8).

[0019] For a further review of PKCs, see Owczarek et al., Cytogenet.Cell Genet. 89: 240-241, 2000 and Hayashi et al., Biochim Biophys ActaMay 6, 1999; 1450(1):99-106.

[0020] Kinase proteins, particularly members of the protein kinase Csubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of kinase proteins. The present invention advances the stateof the art by providing previously unidentified human kinase proteinsthat have homology to members of the protein kinase C subfamily.

SUMMARY OF THE INVENTION

[0021] The present invention is based in part on the identification ofamino acid sequences of human kinase peptides and proteins that arerelated to the protein kinase C subfamily, as well as allelic variantsand other mammalian orthologs thereof. These unique peptide sequences,and nucleic acid sequences that encode these peptides, can be used asmodels for the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate kinase activity incells and tissues that express the kinase. Experimental data as providedin FIG. 1 indicates expression in humans in the lung (including fetaland carcinoid lung tissue), lymph (including mantle cell lymphomas ofthe lymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain(including fetal), heart (including fetal), fetal liver, uterus, andpancreas.

DESCRIPTION OF THE FIGURE SHEETS

[0022]FIG. 1 provides the nucleotide sequence of a cDNA molecule thatencodes the kinase protein of the present invention. (SEQ ID NO: 1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available; that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin humans in the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas.

[0023]FIG. 2 provides the predicted amino acid sequence of the kinase ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0024]FIG. 3 provides genomic sequences that span the gene encoding thekinase protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As indicated in FIG. 3, SNPs were identified at 44 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0025] General Description

[0026] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a kinase protein or part of akinase protein and are related to the protein kinase C subfamily.Utilizing these sequences, additional genomic sequences were assembledand transcript and/or cDNA sequences were isolated and characterized.Based on this analysis, the present invention provides amino acidsequences of human kinase peptides and proteins that are related to theprotein kinase C subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these kinase peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest aft known protein/peptide/domain that hasstructural or sequence homology to the kinase of the present invention.

[0027] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known kinase proteins of theprotein kinase C subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas. The art hasclearly established the commercial importance of members of this familyof proteins and proteins that have expression patterns similar to thatof the present gene. Some of the more specific features of the peptidesof the present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown protein kinase C family or subfamily of kinase proteins.

SPECIFIC EMBODIMENTS

[0028] Peptide Molecules

[0029] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the protein kinase Csubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the kinase peptides of the present invention, kinase peptides,or peptides/proteins of the present invention.

[0030] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the kinase peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0031] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0032] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% ( by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0033] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0034] The isolated kinase peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas. For example, anucleic acid molecule encoding the kinase peptide is cloned into anexpression vector, the expression vector introduced into a host cell andthe protein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

[0035] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0036] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0037] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the kinase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0038] The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the kinase peptide.

[0039] In some uses, the fusion protein does not affect the activity ofthe kinase peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0040] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the kinase peptide.

[0041] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the, peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0042] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the kinase peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0043] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0044] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller(CABIOS,.4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

[0045] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (Nucleic AcidsRes. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0046] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the kinase peptides of the present invention as well as beingencoded by the same genetic locus as the kinase peptide provided herein.The gene encoding the novel kinase protein of the present invention islocated on a genome component that has been mapped to human chromosome19 (as indicated in FIG. 3), which is supported by multiple lines ofevidence, such as STS and BAC map data.

[0047] Allelic variants of a kinase peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. The gene encoding the novel kinase protein of thepresent invention is located on a genome component that has been mappedto human chromosome 19 (as indicated in FIG. 3), which is supported bymultiple lines of evidence, such as STS and BAC map data. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a kinase peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0048]FIG. 3 provides information on SNPs that have been found in thegene encoding the kinase proteins of the present invention. SNPs wereidentified at 44 different nucleotide positions, including anon-synonymous coding SNP at position 42934. The change in the aminoacid sequence that this SNP causes is indicated in FIG. 3 and canreadily be determined using the universal genetic code and the proteinsequence provided in FIG. 2 as a reference. SNPs outside the ORF and inintrons may affect control/regulatory elements.

[0049] Paralogs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a kinasepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0050] Orthologs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a kinase peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0051] Non-naturally occurring variants of the kinase peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0052] Variant kinase peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0053] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0054] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0055] The present invention further provides fragments of the kinasepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0056] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a kinase peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the kinase peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe kinase peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0057] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inkinase peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0058] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0059] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646(1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62(1992)).

[0060] Accordingly, the kinase peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature kinase peptide is fused withanother compound, such as a compound to increase the half-life of thekinase peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature kinase peptide, such as aleader or secretory sequence or a sequence for purification of themature kinase peptide or a pro-protein sequence.

[0061] Protein/Peptide Uses

[0062] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0063] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0064] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that kinase proteins of the present invention areexpressed in humans in the lung (including fetal and carcinoid lungtissue), lymph (including mantle cell lymphomas of the lymph node),ovary tumors, kidney, colon, cervix, bone marrow, brain (includingfetal), heart (including fetal), fetal liver, uterus, and pancreas.Specifically, a virtual northern blot shows expression in lung,carcinoid lung tissue, lymph, mantle cell lymphomas of the lymph node,ovary tumors, kidney, colon, and cervix. In addition, PCR-based tissuescreening panels indicate expression in bone marrow, brain (includingfetal), colon, heart (including fetal), kidney, lung (including fetal),fetal liver, uterus, and pancreas. A large percentage of pharmaceuticalagents are being developed that modulate the activity of kinaseproteins, particularly members of the protein kinase C subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates expression in humansin the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas. Such uses canreadily be determined using the information provided herein, that whichis known in the art, and routine experimentation.

[0065] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to kinases that are related tomembers of the protein kinase C subfamily. Such assays involve any ofthe known kinase functions or activities or properties useful fordiagnosis and treatment of kinase-related conditions that are specificfor the subfamily of kinases that the one of the present inventionbelongs to, particularly in cells and tissues that express the kinase.Experimental data as provided in FIG. 1 indicates that kinase proteinsof the present invention are expressed in humans in the lung (includingfetal and carcinoid lung tissue), lymph (including mantle cell lymphomasof the lymph node), ovary tumors, kidney, colon, cervix, bone marrow,brain (including fetal), heart (including fetal), fetal liver, uterus,and pancreas. Specifically, a virtual northern blot shows expression inlung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymphnode, ovary tumors, kidney, colon, and cervix. In addition, PCR-basedtissue screening panels indicate expression in bone marrow, brain(including fetal), colon, heart (including fetal), kidney, lung(including fetal), fetal liver, uterus, and pancreas.

[0066] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the kinase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in the lung (including fetal andcarcinoid lung tissue), lymph (including mantle cell lymphomas of thelymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain(including fetal), heart (including fetal), fetal liver, uterus, andpancreas. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the kinase protein.

[0067] The polypeptides can be used to identify compounds that modulatekinase activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the kinase.Both the kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

[0068] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the kinase protein and a molecule that normally interacts withthe kinase protein, e.g. a substrate or a component of the signalpathway that the kinase protein normally interacts (for example, anotherkinase). Such assays typically include the steps of combining the kinaseprotein with a candidate compound under conditions that allow the kinaseprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the kinaseprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0069] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0070] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0071] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

[0072] Any of the biological or biochemical functions mediated by thekinase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the kinase can be assayed.Experimental data as provided in FIG. 1 indicates that kinase proteinsof the present invention are expressed in humans in the lung (includingfetal and carcinoid lung tissue), lymph (including mantle cell lymphomasof the lymph node), ovary tumors, kidney, colon, cervix, bone marrow,brain (including fetal), heart (including fetal), fetal liver, uterus,and pancreas. Specifically, a virtual northern blot shows expression inlung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymphnode, ovary tumors, kidney, colon, and cervix. In addition, PCR-basedtissue screening panels indicate expression in bone marrow, brain(including fetal), colon, heart (including fetal), kidney, lung(including fetal), fetal liver, uterus, and pancreas.

[0073] Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

[0074] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the kinase (e.g. binding partners and/or ligands).Thus, a compound is exposed to a kinase polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble kinase polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble kinase polypeptide, itdecreases the amount of complex formed or activity from the kinasetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the kinase.Thus, the soluble polypeptide that competes with the target kinaseregion is designed to contain peptide sequences corresponding to theregion of interest.

[0075] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the kinase protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0076] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of kinase-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a kinase-binding protein and a candidate compound are incubated inthe kinase protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thekinase protein target molecule, or which are reactive with kinaseprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0077] Agents that modulate one of the kinases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0078] Modulators of kinase protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the kinase pathway, by treating cells or tissuesthat express the kinase. Experimental data as provided in FIG. 1indicates expression in humans in the lung (including fetal andcarcinoid lung tissue), lymph (including mantle cell lymphomas of thelymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain(including fetal), heart (including fetal), fetal liver, uterus, andpancreas. These methods of treatment include the steps of administeringa modulator of kinase activity in a pharmaceutical composition to asubject in need of such treatment, the modulator being identified asdescribed herein.

[0079] In yet another aspect of the invention, the kinase proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993), J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

[0080] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

[0081] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0082] The kinase proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the lung (including fetal andcarcinoid lung tissue), lymph (including mantle cell lymphomas of thelymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain(including fetal), heart (including fetal), fetal liver, uterus, andpancreas. The method involves contacting a biological sample with acompound capable of interacting with the kinase protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

[0083] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0084] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0085] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0086] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0087] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the lung (including fetal and carcinoid lungtissue), lymph (including mantle cell lymphomas of the lymph node),ovary tumors, kidney, colon, cervix, bone marrow, brain (includingfetal), heart (including fetal), fetal, liver, uterus, and pancreas:Accordingly, methods for treatment include the use of the kinase proteinor fragments.

[0088] Antibodies

[0089] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0090] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0091] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0092] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0093] Antibodies are preferably prepared from regions or discretefragments of the kinase proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or kinase/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0094] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0095] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0096] Antibody Uses

[0097] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in humans inthe lung (including fetal and carcinoid lung tissue), lymph (includingmantle cell lymphomas of the lymph node), ovary tumors, kidney, colon,cervix, bone marrow, brain (including fetal), heart (including fetal),fetal liver, uterus, and pancreas. Specifically, a virtual northern blotshows expression in lung, carcinoid lung tissue, lymph, mantle celllymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. Inaddition, PCR-based tissue screening panels indicate expression in bonemarrow, brain (including fetal), colon, heart (including fetal), kidney,lung (including fetal), fetal liver, uterus, and pancreas. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0098] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in the lung (including fetal andcarcinoid lung tissue), lymph (including mantle cell lymphomas of thelymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain(including fetal), heart (including fetal), fetal liver, uterus, andpancreas. If a disorder is characterized by a specific mutation in theprotein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

[0099] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas. The diagnosticuses can be applied, not only in genetic testing, but also in monitoringa treatment modality. Accordingly, where treatment is ultimately aimedat correcting expression level or the presence of aberrant sequence andaberrant tissue distribution or developmental expression, antibodiesdirected against the protein or relevant fragments can be used tomonitor therapeutic efficacy.

[0100] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide, digest, and other physical assaysknown to those in the art.

[0101] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in the lung(including fetal and carcinoid lung tissue), lymph (including mantlecell lymphomas of the lymph node), ovary tumors, kidney, colon, cervix,bone marrow, brain (including fetal), heart (including fetal), fetalliver, uterus, and pancreas. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

[0102] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the kinase peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0103] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0104] Nucleic Acid Molecules

[0105] The present invention further provides isolated nucleic acidmolecules that encode a kinase peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the kinase peptides of thepresent invention; an allelic variant thereof, or an ortholog or paralogthereof.

[0106] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0107] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0108] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0109] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0110] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0111] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0112] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0113] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0114] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the kinase peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence, (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0115] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0116] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0117] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0118] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0119] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0120] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel kinase protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 19 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data.

[0121]FIG. 3 provides information on SNPs that have been found in thegene encoding the kinase proteins of the present invention. SNPs wereidentified at 44 different nucleotide positions, including anon-synonymous coding SNP at position 42934. The change in the aminoacid sequence that this SNP causes is indicated in FIG. 3 and canreadily be determined using the universal genetic code and the proteinsequence provided in FIG. 2 as a reference. SNPs outside the ORF and inintrons may affect control/regulatory elements.

[0122] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989),-6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C., followed by one or morewashes in 0.2 ×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0123] Nucleic Acid Molecule Uses

[0124] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As indicated in FIG. 3, SNPs, were identified at 44 different nucleotidepositions.

[0125] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0126] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0127] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0128] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0129] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. The gene encoding the novelkinase protein of the present invention is located on a genome componentthat has been mapped to human chromosome 19 (as indicated in FIG. 3),which is supported by multiple lines of evidence, such as STS and BACmap data.

[0130] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0131] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0132] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0133] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0134] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0135] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that kinase proteins of the present invention are expressed inhumans in the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas. Specifically, avirtual northern blot shows expression in lung, carcinoid lung tissue,lymph, mantle cell lymphomas of the lymph node, ovary tumors, kidney,colon, and cervix. In addition, PCR-based tissue screening panelsindicate expression in bone marrow, brain (including fetal), colon,heart (including fetal), kidney, lung (including fetal), fetal liver,uterus, and pancreas. Accordingly, the probes can be used to detect thepresence of, or to determine levels of, a specific nucleic acid moleculein cells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inkinase protein expression relative to normal results.

[0136] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0137] Probes can be used as apart of a diagnostic test kit foridentifying cells or tissues that express a kinase protein, such as bymeasuring a level of a kinase-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a kinasegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in humans inthe lung (including fetal and carcinoid lung tissue), lymph (includingmantle cell lymphomas of the lymph node), ovary tumors, kidney, colon,cervix, bone marrow, brain (including fetal), heart (including fetal),fetal liver, uterus, and pancreas. Specifically, a virtual northern blotshows expression in lung, carcinoid lung tissue, lymph, mantle celllymphomas of the lymph node, ovary tumors, kidney, colon, and cervix. Inaddition, PCR-based tissue screening panels indicate expression in bonemarrow, brain (including fetal), colon, heart (including fetal), kidney,lung (including fetal), fetal liver, uterus, and pancreas.

[0138] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate kinase nucleic acid expression.

[0139] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the kinase gene, particularly biological and pathologicalprocesses that are mediated by the kinase in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas. The methodtypically includes assaying the ability of the compound to modulate theexpression of the kinase nucleic acid and thus identifying a compoundthat can be used to treat a disorder characterized by undesired kinasenucleic acid expression. The assays can be performed in cell-based andcell-free systems. Cell-based assays include cells naturally expressingthe kinase nucleic acid or recombinant cells genetically engineered toexpress specific nucleic acid sequences.

[0140] The assay for kinase nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the kinase proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0141] Thus, modulators of kinase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of kinase mRNA inthe presence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0142] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate kinase nucleic acid expressionin cells and tissues that express the kinase. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in humans in the lung (including fetal andcarcinoid lung tissue), lymph (including mantle cell lymphomas of thelymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain(including fetal), heart (including fetal), fetal liver, uterus, andpancreas. Specifically, a virtual northern blot shows expression inlung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymphnode, ovary tumors, kidney, colon, and cervix. In addition, PCR-basedtissue screening panels indicate expression in bone marrow, brain(including fetal), colon, heart (including fetal), kidney, lung(including fetal), fetal liver, uterus, and pancreas. Modulationincludes both up-regulation (i.e. activation or agonization) ordown-regulation (suppression or antagonization) or nucleic acidexpression.

[0143] Alternatively, a modulator for kinase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thekinase nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in the lung (including fetal and carcinoid lung tissue), lymph(including mantle cell lymphomas of the lymph node), ovary tumors,kidney, colon, cervix, bone marrow, brain (including fetal), heart(including fetal), fetal liver, uterus, and pancreas.

[0144] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0145] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in kinase nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

[0146] Individuals carrying mutations in the kinase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseproteins of the present invention. SNPs were identified at 44 differentnucleotide positions, including a non-synonymous coding SNP at position42934. The change in the amino acid sequence that this SNP causes isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.SNPs outside the ORF and in introns may affect control/regulatoryelements. The gene encoding the novel kinase protein of the presentinvention is located on a genome component that has been mapped to humanchromosome 19 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0147] Alternatively, mutations in a kinase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0148] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0149] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant kinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0150] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0151] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the kinase gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseproteins of the present invention. SNPs were identified at 44 differentnucleotide positions, including a non-synonymous coding SNP at position42934. The change in the amino acid sequence that this SNP causes isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.SNPs outside the ORF and in introns may affect control/regulatoryelements.

[0152] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0153] The nucleic acid molecules are thus useful as antisenseconstructs to control kinase gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of kinase protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into kinase protein.

[0154] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

[0155] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in kinase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired kinaseprotein to treat the individual.

[0156] The invention also encompasses kits for detecting the presence ofa kinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in humans in the lung (including fetal andcarcinoid lung tissue), lymph (including mantle cell lymphomas of thelymph node), ovary tumors, kidney, colon, cervix, bone marrow, brain(including fetal), heart (including fetal), fetal liver, uterus, andpancreas. Specifically, a virtual northern blot shows expression inlung, carcinoid lung tissue, lymph, mantle cell lymphomas of the lymphnode, ovary tumors, kidney, colon, and cervix. In addition, PCR-basedtissue screening panels indicate expression in bone marrow, brain(including fetal), colon, heart (including fetal), kidney, lung(including fetal), fetal liver, uterus, and pancreas. For example, thekit can comprise reagents such as a labeled or labelable nucleic acid oragent capable of detecting kinase nucleic acid in a biological sample;means for determining the amount of kinase nucleic acid in the sample;and means for comparing the amount of kinase nucleic acid in the samplewith a standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect kinase protein mRNA or DNA.

[0157] Nucleic Acid Arrays

[0158] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0159] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0160] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0161] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0162] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationW095/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0163] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0164] Using such arrays, the present invention provides methods toidentify the expression of the kinase proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the kinasegene of the present invention. FIG. 3 provides information on SNPs thathave been found in the gene encoding the kinase proteins of the presentinvention. SNPs were identified at 44 different nucleotide positions,including a non-synonymous coding SNP at position 42934. The change inthe amino acid sequence that this SNP causes is indicated in FIG. 3 andcan readily be determined using the universal genetic code and theprotein sequence provided in FIG. 2 as a reference. SNPs outside the ORFand in introns may affect control/regulatory elements.

[0165] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol.2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0166] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0167] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0168] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0169] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0170] Vectors/Host Cells

[0171] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0172] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0173] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0174] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0175] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0176] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0177] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0178] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor, Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0179] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0180] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0181] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0182] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89(1990)).

[0183] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0184] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0185] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0186] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0187] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0188] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0189] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0190] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0191] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0192] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0193] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0194] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0195] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0196] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0197] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0198] Uses of Vectors and Host Cells

[0199] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga kinase protein or peptide that can be further purified to producedesired amounts of kinase protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0200] Host cells are also useful for conducting cell-based assaysinvolving the kinase protein or kinase protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native kinase protein is useful forassaying compounds that stimulate or inhibit kinase protein function.

[0201] Host cells are also useful for identifying kinase protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantkinase protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native kinase protein.

[0202] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0203] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0204] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

[0205] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0206] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0207] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0208] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

[0209] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 2637 DNA Homo sapiens 1 atggccaccg ccccctctta tcccgccgggctccctggct ctcccgggcc ggggtctcct 60 ccgccccccg gcggcctaga gctgcagtcgccgccaccgc tactgcccca gatcccggcc 120 ccgggttccg gggtctcctt tcacatccagatcgggctga cccgcgagtt cgtgctgttg 180 cccgccgcct ccgagctggc tcatgtgaagcagctggcct gttccatcgt ggaccagaag 240 ttccctgagt gtggcttcta cggcctttacgacaagatcc tgcttttcaa acatgacccc 300 acgtcggcca acctcctgca gctggtgcgctcgtccggag acatccagga gggcgacctg 360 gtggaggtgg tgctgtcggc ctcggccaccttcgaggact tccagatccg cccgcacgcc 420 ctcacggtgc actcctatcg ggcgcctgccttctgtgatc actgcgggga gatgctcttc 480 ggcctagtgc gccagggcct caagtgcgatggctgcgggc tgaactacca caagcgctgt 540 gccttcagca tccccaacaa ctgtagtggggcccgcaaac ggcgcctgtc atccacgtct 600 ctggccagtg gccactcggt gcgcctcggcacctccgagt ccctgccctg cacggctgaa 660 gagctgagcc gtagcaccac cgaactcctgcctcgccgtc ccccgtcatc ctcttcctcc 720 tcttctgcct catcgtatac gggccgccccattgagctgg acaagatgct gctctccaag 780 gtcaaggtgc cgcacacctt cctcatccacagctatacac ggcccaccgt ttgccaggct 840 tgcaagaaac tcctcaaggg cctcttccggcagggcctgc aatgcaaaga ctgcaagttt 900 aactgtcaca aacgctgcgc cacccgcgtccctaatgact gcctggggga ggcccttatc 960 aatggagatg tgccgatgga ggaggccaccgatttcagcg aggctgacaa gagcgccctc 1020 atggatgagt cagaggactc cggtgtcatccctggctccc actcagagaa tgcgctccac 1080 gccagtgagg aggaggaagg cgagggaggcaaggcccaga gctccctggg gtacatcccc 1140 ctaatgaggg tggtgcaatc ggtgcgacacacgacgcgga aatccagcac cacgctgcgg 1200 gagggttggg tggttcatta cagcaacaaggacacgctga gaaagcggca ctattggcgc 1260 ctggactgca agtgtatcac gctcttccagaacaacacga ccaacagata ctataaggaa 1320 attccgctgt cagaaatcct cacggtggagtccgcccaga acttcagcct tgtgccgccg 1380 ggcaccaacc cacactgctt tgagatcgtcactgccaatg ccacctactt cgtgggcgag 1440 atgcctggcg ggactccggg tgggccaagtgggcaggggg ctgaggccgc ccggggctgg 1500 gagacagcca tccgccaggc cctgatgcccgtcatccttc aggacgcacc cagcgcccca 1560 ggccacgcgc cccacagaca agcttctctgagcatctctg tgtccaacag tcagatccaa 1620 gagaatgtgg acattgccac tgtctaccagatcttccctg acgaagtgct gggctcaggg 1680 cagtttggag tggtctatgg aggaaaacaccggaagacag gccgggacgt ggcagttaag 1740 gtcattgaca aactgcgctt ccctaccaagcaggagagcc agctccggaa tgaagtggcc 1800 attctgcaga gcctgcggca tcccgggatcgtgaacctgg agtgcatgtt cgagacgcct 1860 gagaaagtgt ttgtggtgat ggagaagctgcatggggaca tgttggagat gatcctgtcc 1920 agtgagaagg gccggctgcc tgagcgcctcaccaagttcc tcatcaccca gatcctggtg 1980 gctttgagac accttcactt caagaacattgtccactgtg acttgaaacc agaaaacgtg 2040 ttgctggcat cagcagaccc atttcctcaggtgaagctgt gtgactttgg ctttgctcgc 2100 atcatcggcg agaagtcgtt ccgccgctcagtggtgggca cgccggccta cctggcaccc 2160 gaggtgctgc tcaaccaggg ctacaaccgctcgctggaca tgtggtcagt gggcgtgatc 2220 atgtacgtca gcctcagcgg caccttccctttcaacgagg atgaggacat caatgaccag 2280 atccagaacg ccgccttcat gtacccggccagcccctgga gccacatctc agctggagcc 2340 attgacctca tcaacaacct gctgcaggtgaagatgcgca aacgctacag cgtggacaaa 2400 tctctcagcc acccctggtt acaggagtaccagacgtggc tggacctccg agagctggag 2460 gggaagatgg gagagcgata catcacgcatgagagtgacg acgcgcgctg ggagcagttt 2520 gcagcagagc atccgctgcc tgggtctgggctgcccacgg acagggatct cggtggggcc 2580 tgtccaccac aggaccacga catgcaggggctggcggagc gcatcagtgt tctctga 2637 2 878 PRT Homo sapiens 2 Met Ala ThrAla Pro Ser Tyr Pro Ala Gly Leu Pro Gly Ser Pro Gly 1 5 10 15 Pro GlySer Pro Pro Pro Pro Gly Gly Leu Glu Leu Gln Ser Pro Pro 20 25 30 Pro LeuLeu Pro Gln Ile Pro Ala Pro Gly Ser Gly Val Ser Phe His 35 40 45 Ile GlnIle Gly Leu Thr Arg Glu Phe Val Leu Leu Pro Ala Ala Ser 50 55 60 Glu LeuAla His Val Lys Gln Leu Ala Cys Ser Ile Val Asp Gln Lys 65 70 75 80 PhePro Glu Cys Gly Phe Tyr Gly Leu Tyr Asp Lys Ile Leu Leu Phe 85 90 95 LysHis Asp Pro Thr Ser Ala Asn Leu Leu Gln Leu Val Arg Ser Ser 100 105 110Gly Asp Ile Gln Glu Gly Asp Leu Val Glu Val Val Leu Ser Ala Ser 115 120125 Ala Thr Phe Glu Asp Phe Gln Ile Arg Pro His Ala Leu Thr Val His 130135 140 Ser Tyr Arg Ala Pro Ala Phe Cys Asp His Cys Gly Glu Met Leu Phe145 150 155 160 Gly Leu Val Arg Gln Gly Leu Lys Cys Asp Gly Cys Gly LeuAsn Tyr 165 170 175 His Lys Arg Cys Ala Phe Ser Ile Pro Asn Asn Cys SerGly Ala Arg 180 185 190 Lys Arg Arg Leu Ser Ser Thr Ser Leu Ala Ser GlyHis Ser Val Arg 195 200 205 Leu Gly Thr Ser Glu Ser Leu Pro Cys Thr AlaGlu Glu Leu Ser Arg 210 215 220 Ser Thr Thr Glu Leu Leu Pro Arg Arg ProPro Ser Ser Ser Ser Ser 225 230 235 240 Ser Ser Ala Ser Ser Tyr Thr GlyArg Pro Ile Glu Leu Asp Lys Met 245 250 255 Leu Leu Ser Lys Val Lys ValPro His Thr Phe Leu Ile His Ser Tyr 260 265 270 Thr Arg Pro Thr Val CysGln Ala Cys Lys Lys Leu Leu Lys Gly Leu 275 280 285 Phe Arg Gln Gly LeuGln Cys Lys Asp Cys Lys Phe Asn Cys His Lys 290 295 300 Arg Cys Ala ThrArg Val Pro Asn Asp Cys Leu Gly Glu Ala Leu Ile 305 310 315 320 Asn GlyAsp Val Pro Met Glu Glu Ala Thr Asp Phe Ser Glu Ala Asp 325 330 335 LysSer Ala Leu Met Asp Glu Ser Glu Asp Ser Gly Val Ile Pro Gly 340 345 350Ser His Ser Glu Asn Ala Leu His Ala Ser Glu Glu Glu Glu Gly Glu 355 360365 Gly Gly Lys Ala Gln Ser Ser Leu Gly Tyr Ile Pro Leu Met Arg Val 370375 380 Val Gln Ser Val Arg His Thr Thr Arg Lys Ser Ser Thr Thr Leu Arg385 390 395 400 Glu Gly Trp Val Val His Tyr Ser Asn Lys Asp Thr Leu ArgLys Arg 405 410 415 His Tyr Trp Arg Leu Asp Cys Lys Cys Ile Thr Leu PheGln Asn Asn 420 425 430 Thr Thr Asn Arg Tyr Tyr Lys Glu Ile Pro Leu SerGlu Ile Leu Thr 435 440 445 Val Glu Ser Ala Gln Asn Phe Ser Leu Val ProPro Gly Thr Asn Pro 450 455 460 His Cys Phe Glu Ile Val Thr Ala Asn AlaThr Tyr Phe Val Gly Glu 465 470 475 480 Met Pro Gly Gly Thr Pro Gly GlyPro Ser Gly Gln Gly Ala Glu Ala 485 490 495 Ala Arg Gly Trp Glu Thr AlaIle Arg Gln Ala Leu Met Pro Val Ile 500 505 510 Leu Gln Asp Ala Pro SerAla Pro Gly His Ala Pro His Arg Gln Ala 515 520 525 Ser Leu Ser Ile SerVal Ser Asn Ser Gln Ile Gln Glu Asn Val Asp 530 535 540 Ile Ala Thr ValTyr Gln Ile Phe Pro Asp Glu Val Leu Gly Ser Gly 545 550 555 560 Gln PheGly Val Val Tyr Gly Gly Lys His Arg Lys Thr Gly Arg Asp 565 570 575 ValAla Val Lys Val Ile Asp Lys Leu Arg Phe Pro Thr Lys Gln Glu 580 585 590Ser Gln Leu Arg Asn Glu Val Ala Ile Leu Gln Ser Leu Arg His Pro 595 600605 Gly Ile Val Asn Leu Glu Cys Met Phe Glu Thr Pro Glu Lys Val Phe 610615 620 Val Val Met Glu Lys Leu His Gly Asp Met Leu Glu Met Ile Leu Ser625 630 635 640 Ser Glu Lys Gly Arg Leu Pro Glu Arg Leu Thr Lys Phe LeuIle Thr 645 650 655 Gln Ile Leu Val Ala Leu Arg His Leu His Phe Lys AsnIle Val His 660 665 670 Cys Asp Leu Lys Pro Glu Asn Val Leu Leu Ala SerAla Asp Pro Phe 675 680 685 Pro Gln Val Lys Leu Cys Asp Phe Gly Phe AlaArg Ile Ile Gly Glu 690 695 700 Lys Ser Phe Arg Arg Ser Val Val Gly ThrPro Ala Tyr Leu Ala Pro 705 710 715 720 Glu Val Leu Leu Asn Gln Gly TyrAsn Arg Ser Leu Asp Met Trp Ser 725 730 735 Val Gly Val Ile Met Tyr ValSer Leu Ser Gly Thr Phe Pro Phe Asn 740 745 750 Glu Asp Glu Asp Ile AsnAsp Gln Ile Gln Asn Ala Ala Phe Met Tyr 755 760 765 Pro Ala Ser Pro TrpSer His Ile Ser Ala Gly Ala Ile Asp Leu Ile 770 775 780 Asn Asn Leu LeuGln Val Lys Met Arg Lys Arg Tyr Ser Val Asp Lys 785 790 795 800 Ser LeuSer His Pro Trp Leu Gln Glu Tyr Gln Thr Trp Leu Asp Leu 805 810 815 ArgGlu Leu Glu Gly Lys Met Gly Glu Arg Tyr Ile Thr His Glu Ser 820 825 830Asp Asp Ala Arg Trp Glu Gln Phe Ala Ala Glu His Pro Leu Pro Gly 835 840845 Ser Gly Leu Pro Thr Asp Arg Asp Leu Gly Gly Ala Cys Pro Pro Gln 850855 860 Asp His Asp Met Gln Gly Leu Ala Glu Arg Ile Ser Val Leu 865 870875 3 43950 DNA Homo sapiens 3 cgcggcgggg agggcagggg tgacgctcggagaacagaga ggccgaaccc agagagcggg 60 ccgggacctg ataccgattt cccacccgtcccctgccatg ggcgccggac gcctgccgga 120 gagggctccc ctccttaaag ggccagtggcctccaagccc gacgcctgcg accggcggtg 180 ggtgatagtg tttcccctcc ctgtccagccgagggaaaag ttaactttcc aggcttggct 240 gtgttcaggg aaggaactgg tctcgcctgcctgccctcca tccctcacac catcccttgt 300 cccggaccct ggaggcggag gtccagcccccaactcggag gccccgggcc caccctcccc 360 ttccgccccc ggcccctcgg caggctccgcccctctctga cgtcgccgag gcccgcgccg 420 attggtcgac tgcactgtcg ctccggacacttcctcctgg gccgccgccg ccgccgccga 480 cttaaacttt ggagggggaa aaagagctactggcgcctgg cgaccctccc tgccccccac 540 ccaaccccgc tccggcaacg cccccttcctcacggctccc gaccgaactt ttctccaact 600 tctgcgactc gtgagattcc cttctacccactccggccct cgggacccct ctgcccatcc 660 cctggccggt cgggtccctg cgaacccctttatctctgga atccactcgg tccccgactc 720 agagactcct gccctccacc cccaaggtgaattcccccgg gccgccttct gagtgggatc 780 ctcttcttgg agcactggat cctgggattccctctgcccc cttctcaatc cctcctctag 840 ggaaggggcc tttgaatcgc gggctctcctgatccctgtg accccgacct actagatttc 900 ctctcaggct tcttggaatc tcaatcgctgggacctccaa cccactactt ttctcctttc 960 tgatcttctg ggagccctgg attccgggcctctgacccac tatagtgcct ttctctcctt 1020 cccaggaccc cgccatcctc aggtcccctccgcctgccag atcttttctc ggatccccgc 1080 tctcccacca cctgctcacg agatcccgcggatctagaac ccagggtccc ccggggcccc 1140 ccggccgggt cccgggtggg ctccaggcggccggtccccg gcctcccccc atggccaccg 1200 ccccctctta tcccgccggg ctccctggctctcccgggcc ggggtctcct ccgccccccg 1260 gcggcctaga gctgcagtcg ccgccaccgctactgcccca gatcccggcc ccgggttccg 1320 gggtctcctt tcacatccag atcgggctgacccgcgagtt cgtgctgttg cccgccgcct 1380 ccgagctggc tcatgtgaag cagctggcctgttccatcgt ggaccagaag gtgagggcgc 1440 aggctccctg gatccagctc ggggagaggttgaaggaggg ggcgctggca gaggggtctg 1500 gggcctggtg tgcggaagag ggaggaaggagacctgagct ttgggtgatg gagggatagg 1560 gggcattgcc ccccttccat tgcccctctccccaccatcc ctttgagaga ggactgggca 1620 ggggtggggt gccccagagg cctccccaaatttcatgtcc ctgcatgtcg ttgttttctg 1680 cagcaaacag ggaggaaggg aggggccagccaggtgtaga gaggggagga aggggcagca 1740 gatgtcggcg gacctccacg tccaggcccatcccgggcct cccatttggt ggaaacagga 1800 gaaattgaac ccgggctggc catggtgatccggtgacatg tgtgggtgca ggtgcttgag 1860 ttagctgcca ggggcaagtg aggtctcggagcccaattct gccctcccct aagcctgaga 1920 tatgtgtgga ggggcaggca ctcctacagaccctggggac tctattccct ttcctagtca 1980 cagtgctgtt agcctactct taattttggacaccagggtc cccagggtgg gcagctgggt 2040 gttatggcaa gaggaaacca ggtggaactccacgtctaaa ccgtgaaatg ttaaaagaat 2100 agtgggcttc tgtgttggag tactggactgtagaaatgtt agaatattag aatcataact 2160 tgttggaata tgcatcctag gcaattaaattgcccccatg ttcgtgttca aatattagaa 2220 ttctaggttt gtgaaatagt aaaacattaaaatgctggaa tattagattc ctagattgtt 2280 gaatcctaga aagttaaaat gttagaattttagaatgctg gatggatgag gtccttgaat 2340 gctaaagaat tcaaagagca cagtcctagcttgtcagact cctagaatat taaaatatta 2400 gattaccgct tatttaggtt attgaaatcctaaaatgtat agtgatacca ggtaggaatc 2460 tagaatgtat aattctataa tgtgagcatgttggagtccc aaaatatcca aattccagaa 2520 tcttttcaga ctcctggaaa tgaatcctttgggcatcaga gaaacgtggg gaactgggcc 2580 agctccccca ttctacagac aaggaaactgaagcttagag aaaaacttcc caaggggtca 2640 gggccaaggc agtcctggtc ttctgtggactctctcttag cagtgagaac tgatagggtt 2700 ttgcccacca aatgcctaaa tcccgcaggcccagctcacc accccaactc agcccacttc 2760 atgggaagct ggtggcagtg ggggtacgggggcagattgt cccttgggtg aacttctttg 2820 tccagtgctc aagtccccag cctgccccgctcaggcttca ccccagtttt atttttctgc 2880 caggtccagg tgtgttaggg ccgcgtaccttccttcccga ggccccaccg gggcagtttc 2940 actttctgtt ctactaggtt tcatttcctgcccccaggcc cccaaagctg aggacccaga 3000 cacctgggtc ctttgagcat tgggtggcaggcgccctcct tatctccagc gccctcgagt 3060 ccaagtcccc cggccccccc cccccactttcccaggagcc ccgaaaagtc ctccttccag 3120 ctcgccccac cccagtgctg ggcctggagccaggtaactg ggacaacaat agacagatcc 3180 aggaaggaag ctggggggcg ggtgtgtgagcctggggagg aggcacaggg gagggagtgt 3240 tcattcagca tcccctccca cctccgccaggttccggaaa attcgaggtg tccacgctcc 3300 cggagccact ctccctccca ccccagctcccccttccagc caccaaaccc acgccggcgc 3360 cccctccccg tacaattggg gcgctggcatcctgcccggc tcgcgctggg gttgggaggg 3420 ggcaggcagg aagcgagggc ctgcggggtctctgcgtttc cgggggaaac agccggccct 3480 gccctgggag ggtcacagtc cgcccgctgctgaaggcggc tctgagcttt tccgtcgcca 3540 catccctctc ccgcccctca gttccctgagtgtggcttct acggccttta cgacaagatc 3600 ctgcttttca aacatgaccc cacgtcggccaacctcctgc agctggtgcg ctcgtccgga 3660 gacatccagg agggcgacct ggtggaggtggtgctgtcgg gtgagaggtg gtggccggcc 3720 tgggggcggg gcctcgggtg ggggcggggcatctggggga ggagagggta gggggagtta 3780 gaagtcagga gaggccgggt gtagtggctcacgcctgtga tcccagcact ttgggaggct 3840 gagctggagc tggggggatc gcttgagcccaggagttcga gatcagcctg ggcaacatag 3900 tgagattcca tctctacccc tttctctccctctgaaaaaa aaaaataagg agagttgggg 3960 gcttctggaa gatggttaca gagtggggtcatgaaggcgc tctttaggga ctggtctaaa 4020 ctttcattta tggattagga tgctagtgacacgctttgta cagtttgaaa attcattgag 4080 ctgtgcactt gtgatgtgcg gcctttcctgaacatatgtt atacttattt atttataaaa 4140 ctagtcaagt gcagtagtta gaagggggaaaagaggagaa gaaggagttg gatctgtaac 4200 tgactgtgtt atgcttaaat ataaaggtaaaaaatgggcc agctgcagtg gctcacacct 4260 gtaatcccag cagtttggga ggctgaggtgggaggatcgc tggagcccag gagtttgaga 4320 ccagcctggg caacataagg agaccccatctcttaaaaaa aaaaaaaaaa aaaaaagtta 4380 accgggcgag gtggcacacg tctgtagtctcagctacttg ggaggctgag gtgggaggat 4440 ttcttgagct taggagtttg aggctgcagtgagccacgat catgtcactg cactccagcc 4500 tgggcaacag agagagaccc tatctctaaaaaagaaaaaa agtagaaaaa gaaaaaaaaa 4560 agttatgatg tccatggctc ctgccacgaaaatgctaaat taaatcagaa tctctgcaaa 4620 gtgagatgga atctgcacat cagtatttttaaaagccccc aggtgatttt ctaagacaca 4680 gccagaagcc agttcatcca ctcactattccagtagtata gatgggcatg ctctcagcac 4740 cttagagcag tctatggccc ttggtccctcttgagggtgg gggcagctgc ctttttcatg 4800 gctgtcttcc ctgctgctcc ggcatactgcagtgcccagt gaaaccggct caatgaatga 4860 atgacagaag tctggattta cacctttagtgaccttgttc aggctttaag tactctttca 4920 tatcataagc tggcctcact tgaatttttatcttcattgt tgtctctccc ctaaacctga 4980 gttttgtttt gtttttgtca tttttattattttttgtttt tttagacgga gtctcgctct 5040 gtcacccagg ctggagtgca gtggcgcaaactcagcttgc tgcaacctct gcctcctggg 5100 ttcaagcgat tctcctgcct cagcctcccgagtagctggg attacaggcg cctgctacca 5160 cacgtggcta atttttgtat ttttagtagagacgggattt caccttgttg gccaggctgg 5220 tctcgaactg ctgatcttaa gtgatctgcccacctcagcc tcccaaagtg ctgcgattac 5280 aggtgtgagc caccgctccc ggccctgttattttgttttg aggcagggtc ttgttctgtc 5340 acccaggctg gaatgcagtg gcatgaccaccactcactgc agcctctacc tcccagactg 5400 aagcaatcat cccgcctcag cctcctgaggtggctggact ataggcatta caggcatgca 5460 ccaccacact gggctttttt tttttttcttttttttagac agaatcttac tctgtcaccc 5520 aggctggagt gccgtggcat gatcttggctcacggcaacc tctgcctccc gggttcaagc 5580 aattctcctg cctcagcctc ctgagtagctgggattacag gcacgcggca ccaggcctgg 5640 ctaatttttg tatttttagt agagacggggtttcatcatg ttggccaggc tggtttcgaa 5700 cttctgacct caagtgatcc gcccacctgggcctcccaaa gtgctgggat tacagatgtg 5760 agccaccggg caccgcctat ccatgttcttttttgttgtt ggtggtggta tttttaatta 5820 aaaatttttt aatttggtaa aatatacataacataaaaat tactatttta ggccgggtgc 5880 agtggctcac gcctgtaatc ccaacactttgagagaccga ggcgggcaga tcacctgagt 5940 cgggagtttg agaccatccc tggccaacatggtgaaactc cgtctctact aaaaatacaa 6000 aaattagtcg ggtgtggtgg cgcatgcctgtaatcccagc tactctggag gctgaggcag 6060 gagaactgct tgaacccggg aggcggacttgtggtgagcc gagatctcac tactgtactc 6120 cagcctgggt gacagagtga aactctctaacaaacacaaa caaaaaagcc cacaacattt 6180 taagcacttt taagcgtaca gttcagtaatttaaagttca cgcacactgt tgtgcagccg 6240 gtctccagaa ctgttgtcat cttgcgaaactgaagctcct tgcccgttaa acaactcccc 6300 aattcccgct ctgtccctgc ccagggcgtagggatatatg tgttttgttc aggggtggag 6360 ctgggatttg aacccaggca gaatgtagtatgagagcaaa tgaaggaagg aaggaaagat 6420 cacaccttgc ggctgggagc actgtgagaaatcagggaac gtggggtctg gaaaagcttt 6480 ggcctacccc gcctcaagca tccacccctattttccgcct acagcctcgg ccaccttcga 6540 ggacttccag atccgcccgc acgccctcacggtgcactcc tatcgggcgc ctgccttctg 6600 tgatcactgc ggggagatgc tcttcggcctagtgcgccag ggcctcaagt gcgatggtga 6660 gagctaaagg gttgggggcg gggcctggggcggggctctg caccgggggc ggagcgtaat 6720 ggtcctggca cggggacagc gtggggaggaggagcgggtc tcagagctgg gggcgcagcc 6780 taggaagtaa taatgggaag aaggatgggcccagaagcag agcttgggga aggagtggtg 6840 gggctgggcc ggggctcagg tctaggggcggagcctagga ggtggagctg ggagggacaa 6900 gtaggggctt aagaacagag cctaggggagcagaagggtg gcgggggaag agggtggggc 6960 ctctatcagt tagggatcaa gcagagaaacatccaggagg agatatatat tgagatattt 7020 atatgcaagg aatcagctta cagaattgtgtgggctggct aggcaactca aatctggctg 7080 ggcacagtgg gggaggccag taatcccagcactttgggag gcaaaggtag gtggatcact 7140 tgaggccagg agttcaagac cagcctgggcaacatagcaa gactctgcct gtacaaaaaa 7200 taattagcca agcatggtga cagacacttgtggtcccagc cacttgggag gctgaggcgg 7260 gaggatcact tgagcctggg agctcgacactgtagtgagc cctgattgca ccactgcaca 7320 ccagcctggg tgacagagcg agaccctggctcaaaaacag gaaaaaggcc ggacacggtg 7380 gctcatgcct gtaatcccag cactttgggaggccgaggcg ggtggatcac gaggtcagga 7440 gattgagacc ctccctggct aacatggtgaaaccccgtct ctactaaaaa tacaaaaaat 7500 tagccggacg tggtggcaca cgcctgtagtcccagctact tgggaggctg aggcaggaga 7560 attgcttgga cctgagagga ggaggttgcagtgagccgag attgtgccac tgcactccag 7620 cctggtgata gagtgagact ccttctgaaaacagaaacaa aaacaaaaca ataaaaagaa 7680 aaagaaaaaa aaatccatcc tatcaggaagggcaagtggg aactcaggca caagctgaag 7740 ctgatgtcca caggtggaat ttcttcatccgaaaagtctc tgatctgctt tttaaaacat 7800 tcagctgatt gaatgagacc cacctagaacaagcaggatc acctctccca cttacagtca 7860 gctgattatg gattttcatc acatccagaaaatacctcca ctgggccggg tgcggtggct 7920 cacgcctgta atcccagcac tctgggaggccgaggcaggt gaatcacctg aggtcaggag 7980 ttcgagacca gcctgtccaa catggtgaaaccccgtctct actaaaaata caaaaaagcc 8040 ggcgtgttgg tggacgcctg taattccagctactcgggag gctcagtcag gagaatctct 8100 tgaacccggg aggcagagct tgcagtgagctgagattgca ccattacact ccagcctggg 8160 caacaagagc aaaactctgt ctcaaaaaaatgaaaagaaa agaaaatacc tccatggggc 8220 cttctctccc cagttcttcc tggagtcggggaaaagctgg gttgagaagg tgaaaagaaa 8280 aaacaaacct tgactgggca cagtggttcacacctgtaac cccagcactt tggaggctga 8340 ggcaggcgga tcatgaggtc aagagattgagaccaccctg gccaacatgg tgaaacccca 8400 tctctcctaa aaatacaaaa attagcgggcgtggtggcat gtgcctatag tcccagctac 8460 ttgggaggct gaggtaggag aatcacttgaacccaggaga cagaggttgc agtgagccga 8520 gatcgtgcca ctgcactcca gcctggcaacagagcgagac tccgtctcaa aaaaaaaaaa 8580 acaaaaaaaa aaaacacaaa caaaccaaccttcatggcaa catctagatt agtgtctgaa 8640 taactgtgga tctcgcctag ccaagctgacacattaacat gactatcagg gtccatctct 8700 tgtcaacctg gcacctgtct tagtttgtcagggctgcctt aacaaaatac caccctgcgt 8760 ggcttaaatg acagacattt acttctcaaaatccctggaa ttgtgagagg ctggaaagac 8820 aaagatccag attctggcag ggttctgtttctggtgtagc ctgctttcct gccttgcaga 8880 gggccatcat ttcactgtgc gctcacatgggacacggaga gagagatccc tggtatctct 8940 tccctttata aggaaggcca ggcatggtggctcatgccta taatcccagc actttgggag 9000 gatggtggat cgcttgagtc caggagttcgagaccagcat gggcgacatg gtgaaacccc 9060 gtctctaaaa aatacaacaa attggccaggcatggtggtg catacctcta gtcctagcta 9120 ctcaagaggc tgaggtggga ggatcacctgggcctgggag gttgaggctg cggtgagccg 9180 tgatcatgcc actgcactcc agcctaggtgacagaacacg attgtctcag gaaaaaaaaa 9240 aaaaaaaaaa aaaaaagggt caccagtcccattggattac agccacactc tttcggcctc 9300 aattaacctt aattacctcc ataaaggcaccgtctccaga tatagttgca ttggaggtta 9360 gggtttcaac ataagaattt tgggggagacacagacattt agtccataac agcacccata 9420 catatctcct taaatcatag tttaaaaatatacaggtttt cttttttgga gacagcgtct 9480 cagtctgtca cccaggctgg agtgcagtggcgcgatctca gctcaccaca acctccactt 9540 cccaggctca agcgattctc ctgcctcagcctaccgagta gctgggatta caggcacaca 9600 ccattactgc ccggctaatt tttgtatttctagtagagac ggggtttcac cacgttggcc 9660 aggctggtct tgaactcctg acctcaaatgatccacccgc cttgccctcc cacagtgctg 9720 ggattacagg catgagccac cgcgcctgtccaaaacatac agttctttaa gccaagatgt 9780 ctcaaggttc agcccaagtg tcaagatctatataggtcct ctgtccctgt tattcatgct 9840 tctgagtgag aatgttgaaa tcggggctctgcctacagat gaaggccatg tacctgcatt 9900 ggctatgagg acagatgaca ggtgaggaccatccattctg tgatgagacc ctgtggctcc 9960 atttttttgt gtgtgtgaga cagagtcttgctccgtcacc caggatggag tgcagtggcg 10020 tggtcttggc tcactgcaac ctctacctcctgggttcaag caattctcct gcttcagcct 10080 cccaaatagc tgggattaca ggtgcgcaccaccactcctg gctaattttt gtatttttag 10140 tagacggggt ttcaccatgt tggccaggctggtttcaagt aatccaccct cctcagcctc 10200 cccaagtgct gggattacag acatgagccactgcgctggg ccccatgcgc ctccattttt 10260 gtatggtgtg ccctgcaatt agagccatattcttggatgt tccattgggt attaggtctg 10320 agacagcatc tctagctccg tgggtgccacgcttgtacag aaatcctgat tctgggccag 10380 gcacggtggc tcacacctgt aatcccagcactttgggagg ccaaggcggg cggatcatga 10440 ggtcaggagt tagagaccag cctggccaacatggtgaaac cctgtctcta ctaaaactag 10500 aaaaattagc tgggtgtggt ggcgggtacctataatccca gctactcggg aggctgaggc 10560 aggagaatca tttgaacctg agggggtggaggttgcagtg agccgagatc ataccattgc 10620 actccagcct gggtgacagg gtgagactccgtctcaaaaa aaaaaaaaaa aaagaaatcc 10680 agtttctcca atatcctgtg ttccagatcatcatgcagtc caaagtatac ttgtattatt 10740 taaggactct aggcctgcag atactgattcagtgcattaa aagctcttat aaatattgcc 10800 atcgtccaca caccatatcc aactcttgaggtctcagcat atgcagtctt tgtcatgata 10860 cagccctggt gtcatcaagt cctaatgggttatcagcaca gacttcactg gtgcagcatc 10920 acagatgatg gtcccagttc ctatggtggcaagagaaccc caaatgacta cattccgaca 10980 ggagtttaac tctatcctga gactcattctgagagttata gataagattc tgaaattctg 11040 gaaggcacat gagtgattca aggccaacactgggaaatgg ttcctgtgtg caaagaccat 11100 ttgccctgct gaagctcttc ttgcagggccaacaccgttc tccaagcttg cctccgtgat 11160 tacagcatgc agccaagaca gtgcctacaatgaggaggtg tggaactgga aagcctggag 11220 caggcgggta ccagaagggc tcccaaaggctggaggaaca ttcttcactc cagaatagaa 11280 agcgatcctg gaatcgtttg gaatcactggagatgtatta gagcacacat acagaacgtc 11340 cagtgggaaa cagggagttg agctgatttctccatggatg aggattttaa aagataaaat 11400 aggcagggca cagtggctca tgcctgtaatcccaacactt tgggaggctg aggtgggagg 11460 atcacttgag cccaggagtt caagaccagcctgggcaatg tagcgagacc ccatctctac 11520 aaaaaaataa aaataaaaaa attatctgggcatggtagtg tatgtctgtg gttctggcta 11580 ctcaggaggc tgaggcagga ggattacttgagcccaggag ttgaaggctg cagtgagcta 11640 tgattgtgcc attgtgcttc agccgggggtacagggagat cctgtctcta caaaataaaa 11700 taagacaata agaagtcata cttctgcctagtatggtaca atggacctga gtacaactga 11760 gaactctttt tttttttttg aaactgagtctcgctgtatt gcccaggctg gagtgcagtg 11820 gcgtgatctc agctcactac aacctctgcctcctgggttc aagtgattct cctgcctcag 11880 cctccggagt agctgggatt acaggcgtgtgccactacac ccggctaatt ttgtattttt 11940 agtagagatg gggttttgcc atgttggccagtgtggtctc aaactcctga cctcaagtga 12000 tccgccggcc ttggcctccc aaagtgctgggattacaggc gtgagccacc atgcgtggcc 12060 cacactacta agatttaatc acactacttagggattgcct ggattccagg tctacagaaa 12120 agagaaagtg gggtacaggg ggtgagcagacctggaggga tagtgacctt aggggtgggg 12180 gtgaggagag gcattttctt ttggaaagttggggttgggg aaagaggggg aaccaaaggg 12240 gcctcagaaa aaggaaggtc agggttagaagggggaacag gtgtctctag ggagatggac 12300 aggagttttg gggaggacta gaaggaggtgcttaccatag aggactgggg ctgggtcaga 12360 gctttggcgg ggacttttga ggcatccattgttgcagtgg gaaaaggtgg ggtgtgaggc 12420 gcgttcaggg cctggggggc agatggggtgatgtcggggc tacaagctgg aactaggggt 12480 ggagctttgg agggaacctt tgaggtatcccttgttggag tgggaaaatt ttgggtgtga 12540 ggcgtgttca gggtctgggg gacagatggggtgatggcag ggctacaagc tgaaactggg 12600 gacagagctt tggggggagc ctttgaggtgacccttgttg gagtgagaaa aggggtgtgg 12660 gtgtgttcag ggtctggggg acagatggggtgatggtggg gctacaagct ggaacttggg 12720 gcagaactct aaggaggggt gggcctgaaggggctgatac acttacggat agtagtgcct 12780 tttggaggag atcgtgctgg cggggggtgatgggacagga ccaggtgaga gattgggtgg 12840 aaagggcaca acttctcaag aagagacctaggaggggcag acgccatgtc tcttactctc 12900 tggcgccccc tgcaggctgc gggctgaactaccacaagcg ctgtgccttc agcatcccca 12960 acaactgtag tggggcccgc aaacggcgcctgtcatccac gtctctggcc agtggccact 13020 cggtgcgcct cggcacctcc gagtccctgccctgcacggc tgaagagctg gtgaggagat 13080 gggggatggg acgggttggt ggctaggggggtgacttggc ccaggcatgg ggccaacgca 13140 ctgatgtgtc ccctccattc ttgccaatgacagagccgta gcaccaccga actcctgcct 13200 cgccgtcccc cgtcatcctc ttcctcctcttctgcctcat cgtatacggg ccgccccatt 13260 gagctggaca agatgctgct ctccaaggtcaaggtgccgc acaccttcct catccacagc 13320 tatacacggc ccaccgtttg ccaggcttgcaagaaactcc tcaagggcct cttccggcag 13380 ggcctgcaat gcaaaggtta gctgggcctgtcggggagga cagtacaggg tcagaacctc 13440 cttcccgccc caacctggtc ttgtggcaggacacaaggat ctgagccttg ggaccccagg 13500 gcctcagaag gggagggccc tgaatcctagtgttctggga cctttggaat tctggaatct 13560 tagaacctca gttgtgtgtg tgtgtgtgtgtgtgtgtgtg tgtgtgttgt gttgtttttt 13620 gaagacaggg tgtcactcta tcacccaggctggagtgcag tggcgcaatc acggctcact 13680 gcagcttcaa cctcttgggt tcaagtgatcctcctgcctc agcctcccaa gtagctagga 13740 ctacaggtgg tgccaccaca cccagctaattttcttttct tttttttttt tttgagacgg 13800 agtctcactc tgtcgcccag gctggagtgcagtggtgtga tctcgggctc actgcaaact 13860 ctgcctcctg ggatcaggac attctcctgcctcagcctcc tgagtagctg ggactacagg 13920 cgcccgccac catgcctggc taatttttttgtatttttag tagagacggg gtttcaccat 13980 gttagctagg atggtctcga tctcctgaccttgtgatcca cctgcctcga cctcccaaaa 14040 tgccgggatt acaggcgtga gccaccgcgcctggccacac ccagctaatt tttaaatcat 14100 ttgtagagag aaggtatcac tatattgttcaggctggtct tgaactcctg ggctcaagca 14160 atcctcctac ctcggcctcc caaagtgctgggattacagg tgtgagccac cgcgcccagc 14220 tgaacctcag tctttagaac cttggaatcctagattcata acgtgcttag catggaattc 14280 taaaactgta gaacctgaga attctagaatcagaaccata gcattcaaga attccgaatg 14340 atagaattca gctaaaataa caacagaactttagattaca catcttagat ctcccaagtt 14400 atagactctc agagcatgag aattttggaaccatgggatt tgagggtaat agaaacatag 14460 gcacatcaaa tttgagagtc ttagacgtctagaatcatat aagcttgaaa ccatcgtaac 14520 ctagaatcct ggaaattcta gactcccagaactttgaaca atcaaattct agaatccagc 14580 caggtgtggt ggctcatgca tgtaatctcagcactttggg aggccaaggt aggtggatca 14640 cttgagccta ggagtttaag accagcctgggcaacatggt gaaaccctgt ctctacaaaa 14700 aaaattaaaa attagccagg catggcagcatgcatctgtg gttccagcta cttgggactc 14760 tgaggaggga ggattgcttg agcccaggaggttgaggctg cagtgagcca tgattgtgcc 14820 actgcattcc agcctgggtg acagagcaagaacttgtctc aaaaaaagaa aaaaaaaaat 14880 tctagaacct cagaagccta gatccacataaacttagaaa catccaattc aagaatttac 14940 tggaacaatc aaattctaga atcttagaagcctagagcta aagaagcata gaaacatcaa 15000 attctagaat cttgtatgta tagaatcctagaaccttgga atctgcagat tctggaggta 15060 gagaagccta gaattgtaga accctagaactgtcaaattt tagagtttag atatataaca 15120 ccctaaaatc ttggacatta aagagtcttagaagtgttga ctcatagatg tctagagttc 15180 tagaaacttg gacatcaaac tctgaagccttagaaatacg gaatcaggtc aggggcagta 15240 gctcacacct gtaatcccag cactttgggaggcttaggtg ggtggattgc ttgagcccag 15300 gagttcaaga ccagcttgta caacatggaaagaccccatc tctacaaaaa atacgaaaaa 15360 ttagccaggc atggtagtgc gtgcctgtagtttcagctac tcaggaggct gaggtgggaa 15420 gatcgcttga gcctgggagg cagaggttgcagtgagccga gatggtgcca ttgcacactc 15480 tagtctgggt gacagccaga ctgtttcttaaaaaaaaaaa aaaaaaaaaa aaaccagaat 15540 catagaacct tcataaaata ggtttttagtaaactctaga atcttcgatg tatagtgtcc 15600 ctagaaccgt ggaaacactg aactctacagcaatggttct cgaccagggg ccgttttgct 15660 cctaggggat gtttggcaag ggttggagatggttttgttt ggtacgctgg gatagtgcta 15720 ctggcatcca gtaggtagaa gtcagagatgcagctaaaca tcctacaata cacagagcaa 15780 gtgccctaaa acaaggaatt atcctgggcactgtgttagt gtcacgggtt gaggaaccca 15840 gccctagggt gttcagagtc tggagtcacagcacattaga accaataaca cacacacaca 15900 cacacacaca caagtcgggc gcggtggctcacgcctgtaa tcccagcact tttaggaggc 15960 caaggcaggt ggatcatctg aggtcaggagcgcgaaacca gcctgaccaa catggcgaaa 16020 ccccgtctct actaaaaaca caaaaaaatcagctgggcgt ggtagtgggc gcctgtagtc 16080 ccacgcccag ctaatttttg tatttttagtagagacgagg ttttaccatg tagggcaggc 16140 tggtttcgaa ctcctgacct caaatgatctgctctccccg gcctcccaaa ataccgagat 16200 tacaggcggg agccactgca cccagcagtcgtcgggattt tgagtctagc cctcctactt 16260 aatcaagacc cccccgatgg ttgggaaaactgtggctgaa agtgggaaaa tgaccagggc 16320 agcagcagcc agtgttctta cccagacagcaagagtagac tcttttgagc ctgaggctta 16380 gggtcaaggt tcaagccttc caggtaacctctcttcccct tctcacccgt tcccttgttc 16440 cctgtcctac cagactgcaa gtttaactgtcacaaacgct gcgccacccg cgtccctaat 16500 gactgcctgg gggaggccct tatcaatggaggtgagaggc tggggggatg ctggggagaa 16560 aggggaaggg gcaggactgg gtggagacccctctgatgcc tccgtcccca cagatgtgcc 16620 gatggaggag gccaccgatt tcagcgaggctgacaagagc gccctcatgg atgagtcaga 16680 ggactccggt gtcatccctg gctcccactcagagaatgcg ctccacgcca gtgaggagga 16740 ggaaggcgag ggaggcaagg cccagaggtatacacagaac cctccaagag accctggggg 16800 aagaccctcc tgcacagtga acctcaatttctttttctct acaatgggct gacatcacct 16860 catatttata aattttccca gttcctgaggcaaacctttt aaagcactac aatttttttt 16920 aaataatttt ttgtttgaga cagggtctcggtctgtcgcc caggctggtg cagtggtgca 16980 gtcttgactc actgcagcct cgaccacctgggctcaagcg atcctgccac cttagcctct 17040 cgagtagctg ggaccacagg ctcgtccaccacacccagct aatttttgta tttctgtaga 17100 gacagggtct accctatgtt gcccaggctggtcttgaact cctgactcct gagctcaagt 17160 gatccacccg cctcagcctc ccaaagggtcttgctttgtt gcccactgga gtgcagtggt 17220 gtgattgtgg ctcactgtaa cctcaaactcctgggctcag gtgatcctcc tgcctcagcc 17280 tcccgagtat ctgggactac agggatgcactgctatccct ggctaatttt agacggcgtt 17340 tcgctcttgt tgcccaggct ggagtgcagtgatgcaattt cagttcattg caacctctgt 17400 ctcctgggtt caagcgattc tcctgcctcagcctcccaag tagctgggac tacaggcacc 17460 cgcccaggcc cagctacttt ttttgtatttttagtagaga cagggttttg ccatgttggt 17520 caggctggtc ttgaactccc aacctcaggtaatccacctg cctcggcctc ccaaagtgct 17580 gggattacag gcatgagcca ccgcgcctgacctatattcc tcttcttttt tttttttttt 17640 tttttttaag atagggggtc ttgctatgttgcccagggtg gtcttgaact tctgcgctca 17700 agcaatcctc ccacctcagc ctcccaaagttctgggatta caggtgtgtg ccactgtgcc 17760 cccagcctac acatttttaa actatacacggagttcatac ttagtcagct ccactggaat 17820 gtgagctcag gtgcatgagg gcaaggatattttctgccct cccaggtgcc taggacagga 17880 ctggctcaga tcaggcactt cctatctgggtgtggcgtga atgtttattg agaaagcaca 17940 gttcacacag gcgctggagg gtgacagcccagatcccagc tctaccactt cacttgctag 18000 gcgcttccct gtgtgccacg gtttcctcctggggcgatga ggtacctacc ccacggggtg 18060 ataaacctgg ggtaggggta agggggcaccctcacaggtg cactggaaaa tatttaatga 18120 gcacctgctg tgttcaagca cacagctatgaacaaaagag gtaaaagtct gcccttctgg 18180 agctgactgc ctcagtgggg agacagctaataaatgcatc catagcatcg ggtattggta 18240 atggtgataa aaacaagagg agatggagaatgggggacat gctatcttag ggtccttcaa 18300 ggagacctcg ctgaggaagt ggcagttgaagggaggggag ggaaggagcc ttgtggggct 18360 ctgggggaaa aggcttccag gcagaggcaacagcgagtgc aaaggccctg gggtggaggc 18420 accgtgttcc agggacagca aagagacccatgtagctgca gcagggaggg cgaggggaag 18480 agggttggac agaaagggga tgggtaagccagtcacagtg acgacagagt gtttcctgcg 18540 gtgcctccca acccaagcag cctgaagccgcaggttccct ttctcccacg tctttcctgg 18600 gaatgcctag taacaccgtc atacactgtcaagagttgga ccttgaggga ttgggggtgg 18660 cgggtgtggg gagaggcagc ccatttcacagatggggaaa ctgagtctca ggcaaagaga 18720 tgtgatcaag gccacccagg ttctgatctagcacagggat ccagagattg ttggttccag 18780 agttgagcaa gtcacttaat ctctcaaatctcaaactcct gacctcaagt gatcccccca 18840 cttctgcctc ccaaagtgtt gggattacaggcatgagcca ccatgcccag caggccactt 18900 aatctctgta gaccttcctt actgtactaacagcatctgc acaaatgagg gaggtgaggc 18960 ccagagaggt tgaatcactt acccagtgtcacacagctgg ctccacaatt gctggactaa 19020 ataccaatta gcacttactg gaggtcctctgtatgccagg cactgtacta agctccgtag 19080 aaaggtttcc attcctcata gcatcccctttgggtggaca aactgaggca tgaagaggtt 19140 aggtaatttg ctaggcagcc tgacttcagaaaggcctact acagaagccc tctcaagaat 19200 ctccttctgg gccagcgtgg tggctcacacctgtaagcac tctgggaggc cgaggcggat 19260 ggatctcgtg aacggattct aagggtgggactaggggcag gagttaggga aggagttgag 19320 gcaaagagtt cgagaccagc ctggccaacatggtgaaacc tcatcactac taaaaataca 19380 aaaattagcc aggggtggtg gcgtgcacctaatggtcacc gtgattgtcc cggccactca 19440 ggaggctgag gcacgagaat cgcttgaacccgggaggcag aggttgcagt gagccgagat 19500 cgcaccactg cattccagcc tgggtgacagagcgagcctc ttaaaaacaa acaaaaagca 19560 actcccgggt gtgtgttggg gggaaaatgtcaaaacaaac caaacaaaca aaaacagtcc 19620 ccaactccct agtttcccag agatgccccctgcattccca agcagcatgg tcactttctg 19680 catgtgactt ctcacccctt cctcttccttcgcagctccc tggggtacat ccccctaatg 19740 agggtggtgc aatcggtgcg acacacgacgcggaaatcca gcaccacgct gcgggagggt 19800 tgggtggttc attacagcaa caaggacacgctggtgagtg gccggggcgg ggccgggtac 19860 ggcggagcga aggctggaag aggggcggctcagcttgagt aggcggggct aggtgggtgg 19920 ggctggagct aggcgcgagc ggggccagtagtgggctggg ccgtgctgga ggcggggcta 19980 gaattagaag tgtgggctgt aagggtgggactacgggcag gagttaggga agacccgggg 20040 ctcagggcaa ggtcaggggc ggggctagagttaggggagg agcttggctg gaggaagagg 20100 gctaagtggg ggcgagtctg gggttagggcgtgggggctg ggctagggtt aaggctaggg 20160 gcggggctgg ggttagggcg tgtggtggggtggggttacg gcgtggggta ggtgctagag 20220 ttacggcgtg cacgtggtgc tccaggcacctggagcccca agcagctcca cgggataggg 20280 actgggcagg aaagtctggc ggttcacgtgactcttcaaa catctctgca gagaaagcgg 20340 cactattggc gcctggactg caagtgtatcacgctcttcc agaacaacac gaccaacaga 20400 tactataagg taagcctccg ggctttcagctccctcggac ttcccgctgt gcccacaaac 20460 tttcccacac ctcctcctac ccccagttactccagacaga tcctgcaaat cacaccctct 20520 gcccaccccc agcctccctg cttccagctcatcagcaagt gctgcccatc cgattctggc 20580 cccaccactt tccagccagg gggactccgggcaggttccc ttacttctca gtgcctcacg 20640 cttctcacct gcaaaatgcc tcaaatgctaatactcacct cagggctggt gcgagaattc 20700 aaagagccaa tccactaaac caattggcttaaggcgtggt atatattaag ctcccagtaa 20760 ttctaaggct gttctcacta ttcctttattttttgttatt tatttatttt ttgagacaga 20820 gtctcactct gtcgcccagc tggagtgcagtggcgcgatc tcggctcact gcaacctccg 20880 cttcccgggt tcaagcgatt ctcctgcctcagcctcccac cctaggacta caggtgaatg 20940 ccaccacacc cagctaattt ttgtatttttagtagagacg gggtttcacc atgttggaca 21000 ggatggtctt gatctcttga cctcatgatctgcccccctc ggcctcccaa agtgctggga 21060 ttacaggcat gagccaccgc acccggcctcactatttctt tataattaat gtattgcatt 21120 gtgtgcgtat tcgtcaccac ctcccatgcccacactgtgt cccagccact gtcttccacc 21180 tggatggttt cagccttctc cttgcagggtccttgcttct gacctcacaa cctctgtcat 21240 ttcccccaca gccaggggga gtcttcattaaaaccgtcaa accccccagt ggctcccatt 21300 gtcttagaag taataaaacc tggtactccagctgttacct gccctggaag cgtcttcctt 21360 gaactttcca tggctggttc cttatcatcttcccattttg ctcagaccac accatctaaa 21420 atgctgtcct tggccaggcg tggtggctcacgcctgtaat cccagcgctt tcagaggccg 21480 aggtgggcgg atcacttgag atcatgagttcgaaaccagc ctggccaata tggtgaaacc 21540 ttgtctgtac taaaaataca aaaattagctgggcatggtg gcgggtgcct ataaccccag 21600 ctacttggga ggctgaggca ggagaattgcttgaacctgg gaggtggagg ttgcagtgag 21660 ctgagatcgc gtcactgcac tcctgcctgggcaacagagc aagactccat ctcaaaaaaa 21720 taaaataaaa taaaatataa tgctgtcctcaccatgcccc cccgacgtgt ccatgtcatc 21780 acctggtttt atgggctgcc taagtcattcattctttcct ctctcctacc tccctccttc 21840 ctcttttgac acgtttccca ccccatagtccctgtgcctt ctgtcccgcc tgggtcccct 21900 cagcctcctt cctggttctc tgtctccatctcattctatt ccatctgccc tccgcacaca 21960 agcggatgat gctcaaaagc cttcagtggcttcctagggc ccttggacaa agcccaggct 22020 cttccttgtg gcccgcaaag ccctgtgtggcctcatttcc tccatttatt atcaaacgtt 22080 tatttttgag acggagtctc gctctgtcacccaggctgga gtgcagtggc gcgatcttgg 22140 ctcactgcaa cctccgcctc cggggttcaagtgattcttc tgcctcagcc tcccaagtag 22200 ctaggattat aggtgtgcca ccacgcctggctaatttttg tatttttagt agagatgggc 22260 tttcaccatg ttggtcaggc gggtctcgaactcctgactt tgtgatccgc ctgccttggc 22320 ctcccaaagt gttgggatta caggcatgagccaccatgcc cagcccattt atttattttg 22380 agacaggctc ttgccctgtc tcccaggtgcagtggcatga tcatggctca ctgtaacctc 22440 tgcctccctg gctcaaatga ttctcccacctccacagtag ctgggattac aggtgcgcac 22500 caccacacct ggctagtttt tttattttttgtagagatgg gggtctcatt gtgttgctct 22560 ggctggtctc aaactcctgg gctccagcgatctgcctgcc ttggcctccc aaagtgctgg 22620 gattacaggc ttgtggcacc atgcctaatttttaaatttt ttgtagagct ggggtctcac 22680 tgtgttgccc aggctggtct tgaactcctgggccatctgc ccacctcggc ctcccaaagt 22740 gctgggagta caggcacgag ccaccacatccggccatcaa aatgtttatc aagcttttac 22800 tatgtccagg caccgcccca tgtgatggggatacagcttg gcttttgagc atagcctttc 22860 cttagggcct ttgcacatgc tgttcccctactcccttgcc aactggctgc ttcttacctt 22920 tctggtctct gcttcaatat cacttctgccagtaattagt attattatta ttatttttga 22980 gacggaatct cactctgtcg cccaggctggagtgcagtgg tgcgatcttg gctcattaca 23040 accaccgcct cccaggtgca agcgattttcctgcctcagc ctcccgatta gctgggatta 23100 caggcgcaca ccaccacgcc tggctaatttttgtattttc agtagagacg ggattttgcc 23160 atgttggcca ggctggtctc gaactcctgacctcaagtga gctgcccacc tcggccttcc 23220 aaagtgttgg gattacaggc atgagccaccgcacctggcc tctgccagta attataaaag 23280 aacagtgaga acaggcttag aattactgggaacttgtctg accactgtgc aaaccaggcc 23340 catccctatc aacatggatc ccgtgtatccttctgggtaa gcactagaat tccaaggtct 23400 gcctggcatc ctcacctgtg ctggttccacgtcctgcagg aaattccgct gtcagaaatc 23460 ctcacggtgg agtccgccca gaacttcagccttgtgccgc cgggcaccaa cccacactgc 23520 tttgagatcg tcactgccaa tgccacctacttcgtgggcg agatgcctgg cgggactccg 23580 ggtgggccaa gtgggcaggg ggctgaggccgcccggggct gggagacagc catccgccag 23640 gccctgatgc ccgtcatcct tcaggacgcacccagcgccc caggccacgc gccccacagt 23700 aagtcctccc acctcgggtc cttgagagaatagatctaga tgggtggggc acggttctgg 23760 ggaatggaag ggccaaagag gaaagtgggcaatggtgggg ttgagaacgc agcttctgga 23820 ctcagcaggc ctgggttcaa actctgttaatcactcctgt taatcccagc gctttgggaa 23880 gccaaggagg gaggatcact tgaggccaggagttcaagac cagcctgggc aacataatga 23940 gattccatct ctacaaaaaa taaaaacaattagccaggtg tggtggtgca cacctgtagt 24000 tccaggtact tggaaggctg aggcaggagaattgcttgag cctgggagta gtgagtcatg 24060 attgcatcac tgcactccag tctgggtgacagagcaagac tctgtctcca aaacagaaaa 24120 aacaacaaca acaaaaatcc acaacaaatctctgttaagc tcctggcctg atatgtggcc 24180 ctgggcatat cacttcccct ccatgagccttgtcccaggt gctgataagt cctcatgcac 24240 ttactgagtg cctcctctgt gcgggacagtgctggggacc cagtggtggc caggacagcc 24300 caagacctgc cctcatgggg ctcagagtccagtagggcag aatacccatc ttcagagagt 24360 gacagtccag ggtgggcagg gttgggacaaggaagctagg gagctggagg agcccagagg 24420 ggtacctgac ccaatctggg tatataggggggcttcctgg aggaggtgac atctgaactg 24480 agatctggag gccgaggcag ggtgagatgtgggaaagaaa atgggaggtc attttaggca 24540 gaggcaaaaa atgttgagag agtaccaggttcccaccctc tggagcttat aatccagtgt 24600 gggtgacaga cattgatcat taacccatacaagcaacgag tgtgatgcag agcatttgcg 24660 agagtaatcc aacttggtcc taggagtgacatttgagctt acacttgagg atgaggagga 24720 tttagctaag tctaggatga aggaaagagtattcctggca ggggaaacag catatgcaga 24780 gaccagaagg cagaagagag tttgctgtatttgaggccga gcaaggaggc cagtgtgtca 24840 ggaatagcat gttgggggta gaagtcagaggtagatgagg gtctaggcca tggcttttag 24900 gccatttaag gggctcaggc ttcttcctgagggcactggg gagccatggc agagttgtga 24960 gcagaggagg gacagggtca gtcttgtgcctcagtaagat ccctctggtt tctctgtggg 25020 aggtgagtag gaaggggcag gattggggcaaggagaccag ggaaggggct gtggggtgag 25080 gacccagagt tggggggcga gcaggggcctagactggtgg aagagagaga cattcaaatg 25140 gcagaaggat cggactttag aaatgtctggctctggttgg gtttgtaggg ggaaaagttc 25200 aagggaagat gcaggagtca gtctgggctttccctccaag actcagtttc cttctctgta 25260 caatggggtc agtctgcctc ccctggtgctgagatcctgg ggtaaaatgc tcagcaaaat 25320 catctgtaac atcactcctt tagccactcagcacatctca tttactcctc ctggtggctc 25380 tatgagggag gtccttttat tattcccattttctagatga ggaaactgag gttcgtagtg 25440 gacaagtcac cagcctgaag ttgcacattgtatcgaacat tggattcaaa tctgggtggc 25500 ctgactccca agtctgcttt tgcaggtatgggtggagata atcctgagcc tggagtcccc 25560 tcacctctgt ctctcccctc tccctaggacaagcttctct gagcatctct gtgtccaaca 25620 gtcagatcca agagaatgtg gtgagacttcctgcccccac ctgatgccct cccctcccac 25680 aaaccctcct cagctctctc gtctccttgactcccccttc cccatttcca tttgcacccc 25740 tgacctgccc tgtcttcacc ctgtaggacattgccactgt ctaccagatc ttccctgacg 25800 aagtgctggg ctcagggcag tttggagtggtctatggagg tgaggacact tcagagctaa 25860 cccagaggga gccccgggct gggggaagctgctgtggctc cagccctttc tttctggctc 25920 caacccttcc tttctgattg gtcacatgctcacctcccat gttgattggc ttagctagat 25980 cctgggtgga ctgattgcag gttctccttttctcattggg aaaaaccaat ggacattcct 26040 cctgttatta ataggaaggg taaattcggcactctgattg gtcacagagg tagattttga 26100 ttggataggg aaggtagatt ctgcactctgattgaccaca gagctagaac ctagattctg 26160 attggataga gtagattctg cattcatattggccacagaa ctagttccta gattctgatt 26220 ggaaaagagg gtagattctg cactctggccacagagctag atcctagatt ctgattgaat 26280 aggagggtag attctgcatt ctgattggccacaggtctag atcctagatt ctgattggat 26340 tggagggtag attctgcatt ctgattggccacaggctaaa tcctagattc tgattgtatg 26400 gggcgggtgg taaattttac actttgatttgccacagagc tagatcctag agttcaatag 26460 gacagggagg gtaacttcta cactctaaactctaagactc agtttccttc tctgtataat 26520 agggtcagtc tgcctcccct ggtgctggtgtctctcccct gtccccagga ctcttatggg 26580 tcacacaaaa ctagatgcta gattccgactggttataaat ccagtttccc atgttataca 26640 ttcccttctt cggagctttt tgtttgttttttgctttcct tctttctgcc tttactccca 26700 aggtgcacct caggtggcct tttcacgtatctcctggggc cttccaactc tgcccaactc 26760 tggctgtctc catggtgggg ggcagaggttggcagaggtg gagatactcc tgccaggact 26820 gggtggtctt gctctctcat cccccatctcttctactccc tgtgcaggaa aacaccggaa 26880 gacaggccgg gacgtggcag ttaaggtcattgacaaactg cgcttcccta ccaagcagga 26940 gagccagctc cggaatgaag tggccattctgcaggtaacc accaggccgc cttccctttc 27000 tgcttcttcc tttcatgggc cagctgacccagtgtagggg tggtcaggga aggcttcctg 27060 ggggagggca tgtgcatgtt gagactgaaggggagaaggt gttcttagca gagggaccag 27120 cctgtacaaa gacctggtga gagggagcatgaggttttct agaaaggagg tactgggaga 27180 tgaggccagg gaggagggcg gagccagaccctttggactt tctcctgagg gtactggaga 27240 gccacagaag gcttttgagc aagggaggggcaggatcagg tgtgtacgtt aggaaaatcc 27300 cgcaggctgc catctggagg gtgggtggaaagggaagtga ttgtagccag gaggctgagt 27360 ggggatctgg gtgggagaga ggggttaggccaggatagga ctggagaatg tgagaggggg 27420 tatggattta aaagatacag atgtgcagagctctccccat ttctccaagc tccccctcct 27480 ccctcctgca accctgggcc tccaccagaatttcaggatg taaagatcct tctgggccgg 27540 gcatggtggc tcacgcctgt aatcccagcactttgggagg ctgaggtggg aggatcactt 27600 gaggccagaa gtttgagacc agcctggccaacatggcgaa accccatctc tatatttaaa 27660 tagaaagaaa aaaaagatcc ttctgggcacctggcaggtg gggtggaggt gggcctgttc 27720 tgtcttggcc tgtgggaagc ccccttccctctccaagtgc caatacccca gggacatcct 27780 tctccttgtt tgtcatcctc ctgctcctatacctgacccg ttggggtctg agtttgtggg 27840 ttacctgggc cctgaccccg ctccccaccctgcagagcct gcggcatccc gggatcgtga 27900 acctggagtg catgttcgag acgcctgagaaagtgtttgt ggtgatggag aagctgcatg 27960 gggacatgtt ggagatgatc ctgtccagtgagaagggccg gctgcctgag cgcctcacca 28020 agttcctcat cacccaggtg cgtctgccctgcccgctgcc acccgcccct ccccatcagg 28080 tgtcagcttg gagaggccct gtatgcctagggggtcaagc agacacttgg gggagtcaca 28140 atagcagata acagaaacca tcatcaggctgggcgcagtg gctcacaccc gtaatcccag 28200 cactttggga ggcccacgag gtcaggagatcgaaaccatc ctggctaaca tggtgaaacc 28260 ctgtctctac tagaaataca aaaaattagccgggcatggt ggcaggcgcc tgtagtccca 28320 gctactcggg aggctgaggc aggagaatggtgtgaacctg ggagatggag cttgcagtga 28380 gccgagatcg cgccactgca ctccagcccgggcgacagag caagactcca tctcaagaaa 28440 aaaaaaaaaa aaaaaaagga accataatcgtacagaagta ataataacca taatagaaaa 28500 aataagccgg gcatggtagc acgtgtctgtggtctcagct actcaggagg ctgaggcagg 28560 aggatcactt gatcccagga gttctgtgctgatcaggtgt cctcattaag tttggcatcc 28620 atgtggtgac ctcccaggag tgggggaccaccaggttgca aagcagccca ggttggaaat 28680 ggagcaggtc aaagctctct tactgatcagtagtgggatc acatctgtga agaggcattg 28740 cactccagcc tgggcaacat agcgagaccccgcctctaaa aagaaagaaa gaaaaaagaa 28800 aaataatagt gacaataaca attaaaaataaagagtatgc caggcgcggt ggctcacgcc 28860 tgtaatccca acactttggg aggccaaggcgggtggatca cctgaggtca ggagtttgag 28920 accagcctgg ccaacatggt gaaaccctgtctctactaaa aatacaaaaa ttagctgagc 28980 atggtggcag gcacctgtaa tcatagctacttgggaggct gaggcaggag aatcccttga 29040 gcccaggagg cagaggttac agtgagctgagatcgtgcca ttgtactcca gcctggggga 29100 caagagtgaa acttcgtctc aaaaaaaaaaaaaataataa taataataat aaagagtaat 29160 cataataata gaaaaaaata gactagcggtaatgatagct atttttatta taaaaaataa 29220 atgatcagtc aggctccctg gacctgacttgactcatcta gaaaaaaggg gagtcaggca 29280 tggtggggta cacctgtaat cccagctactcaggaagcta aggccagagg attgcttaag 29340 cccaggagtt tgagccagcc tgggcaacatagcaagagcc catctcaaaa acaggctggc 29400 tcatgcccgt aatcccagcg ctttgggaggccaaggcaag aggatcgctt gaagccagga 29460 gttggagacc agcctaggcg acatagtgagatcccacctc tacaaaaagt aaaaaaaaaa 29520 atagaaaacc tagctggatg tggtgcctggtagcacatgt ctgtagtcct agctgcttgg 29580 gaggaaggga gtggagaggc tctcttgaacctaggtggtt gaggctgcag tgagctatga 29640 ccgtgccact gcactccagc ctgggtgacagagcgagacc gtgtctcaaa accaaacaat 29700 agaaaaaacg ggcaagcagc cctttttctctcattcattc attcagttgg tcaacaaaca 29760 ctccctagtc cctgctctgt gcttggtcccttgctggtca gtgttgagga cacagggatg 29820 accaatacag ccccattctt agacagtgatagctcaggtg agcagggcta ggacaaggga 29880 ggctgataat ggtgatgata aataatgtggtcactaacat ttattgagca cttactatgt 29940 gccaagcact cttcaaactc atttaatcttcatagtaacc tgtgcagtag gtgctattat 30000 tatcaatccc cttttatggt tgaagaaactgagggtcaga gacatcaaat atcttgtcca 30060 gggtcacata gctggtggga tttgaacctaggatctttgc ttttaactag tgatgtcaaa 30120 ctcatttgtg ttacattcaa acagattttccttgtgtgcc tgtgtgcctg tgctttttgt 30180 ttgttttttt gagacagggt ctcgctctgtcacccgagct ggagtgcagt ggtacaatca 30240 tggctcactg cagccttgac ctcccgggttcaagcaattc tcctgcctca gcctcctgag 30300 tagctgagac aacaggcatc agccatcacacccagctaat ttttataaag acatttttat 30360 aaagacttgc tatgttgccc aggctggtcttcaactcctg ggctcaagtg atcctcctga 30420 ctcggcctca gcctcgcaaa gttctgggattacaggtgtg agccactgtg cccggcctct 30480 gttctgcgtt tctttttttt tggtggaggtgcacattaga ttcttatcac ttatattgtt 30540 caatggtttt atcccagtgt ttgcctctttattttatatt tagtttttat ttaccatagg 30600 gttttattta ttttattttt tatttttttttgagacggag tcttgctcta ttgcccaggc 30660 tggagtgcag tggcaccatc tcggctcactgcaagctccg cctcccaggt tcacaccatt 30720 ctcctacctc agcctcccaa gtagctgggactacaggtgc ccaccaccac gcccggctaa 30780 ttttttgtat tttcagtaga gacagggtctcactgtgtta accaggatgg tctcgatctc 30840 ctgacctcgt gatccacccg cctcggcctcccaaagtgct gggattacag gtgtgagcca 30900 ccgcgcctgg cctattttat ttttttttttgagacagggt ctcattttgt cacccaggct 30960 ggagtgcagt ggtgtaatca tagttcactgcagcctcaaa ctcctaggct gaagcaattc 31020 tcctatctca gcctcctgag ttaactggaaccacaggcat gagccaccac gtccagctaa 31080 tttttttttt tttttttttt aatgtttttgtagagacaag gtctcgccat gttgcccagg 31140 ctggtcttga actcctgggc tcgagcgatcctcccatctc agtctcctga gttagctgga 31200 accacaggca tgagccatta cacctggctaattttttttt atgtttttgt agagacaggg 31260 tcttgccatg ttgggtctcg aactcctgggcttaagtggt cctcttgctg cagcctccca 31320 aagttctggg ttacaggcat gagccactgcgtccagccgg ccatagagtg gaacttttac 31380 gatgttaaat atccccttgt gtggtttctgtgtttcacat ccttcctaga aaggcttcct 31440 tctggtgggt gccttgcctt cttctgagacatctctgtgg gtctcagagc catcgttgct 31500 gtgttccctt taccctggcc cagcacccttatcctctcag gcagtgtgcc tgtgtttgtc 31560 aggctggctt atggggtggg gacagaaacccactgatgca ccctcatcca gactttatta 31620 tttatgtatt tttgagacag agtctcgctttgttgcccag gctggagcgc agtgacacga 31680 tctcggctca ctgcaccctc tgccccctgggttcaggtga ttctcctacc tcagcctccc 31740 gagtagctgg gattataggt gtgtgccaccatgcctggct aatttttgta attttagtag 31800 agatggggtt tcatcatgtt gcccaggccagtctcaaact cctgacctca agtcatctgc 31860 ctgcctcagc ctcctgaagt gctgggattacaggcatgag ccatcgtgcc cggccacatc 31920 cagacttcag gtgtggaaag gaatcatggttctcacaggt ggctgctttc agcagctgag 31980 ggggtttctc tttctggcct tcatctcttcctctcttttt gcctgctcgc tcttctttct 32040 ctctctctct ctctctgcag atttctgctttctgggctct tgcctgcccc acacctaagc 32100 cctgtgctaa gccctttacc tcctgagcttatgtaggcct caccaccatc ctaggaggta 32160 ggtattgtta taaaccccat tttatagatgaggaaactga ggctcaggga gttagcagtc 32220 tccctcgagg tcacagccaa gtagctttccagccaagatt tgagtctgga tctatctagc 32280 ttccaacctg ccctctttct tttctttttttttttttttt tttgagacga agtctcactc 32340 tgtcacccag gctggagtgc aatagtacagtctcagctca ctgcaacctc tgcctcccag 32400 gttcaaacaa ttgtcccacc tcagcctcctgagtagctgg gactacaggt gcgtcccagt 32460 acaccgggct aatttttgta tttttagtagagacggggtt tcactatgtt ggccaggcta 32520 gtcttgaact tctgacctcg tgatccacccgcctcagcct cccaaaatgc tgggattaca 32580 ggcgtgagcc accatatccg gccaatgttttttttttttg gagatggagt ctcgctctgt 32640 tgcccaggct ggagtgcagt ggcgctatctcagctcactg caacctctgc ctcccaggtt 32700 caaatgattc tcctgcctca gcctcctgagtagctgggaa cacaggcaca cgccaccatt 32760 cctggctgat ttttgtattt ttagtagagatggggtttca ccatgtcgat caggctggtc 32820 ttgaactttt gatctcgtga tctgcccgcctcagcctccc aaagtgctgg ggattacagg 32880 cgtaagccac cgtgcccggc ctaacctgccctctttgttc acatgaactg ggagaaaatc 32940 aactgacaaa atctggaaat gggcggggcgaggtggctca cgcctgtcat cctagaactt 33000 tgggaggcca aggcagatgg atcacctgaggtcaggagtt ttgagaccag cctggccaac 33060 atggtgaaat cccatcttta ctaataatacaaaaattagc caggtgtggt ggcattcacc 33120 tgtaatccca gctactgggg aggctgaggcacaagaattg cttgaacctg ggaggtggaa 33180 tttgtggtga gtcgaggtca tgccgttgcactccagcgtg ggcaacagag tgagactcca 33240 tctcaaaaaa acaatctgga gatgacatatacaacacatg catctttcca gcttggtctc 33300 ccagtctgta gaatgaggag gttggtcaggcatggtgggt cgtgcctatt atctcaaggt 33360 ttgggtagct gaggtgggaa gatcatttgaggccaggagt tttagaccag cctgggcaac 33420 atagcgagat gccatctcta caaaaagatttttttaaaaa agaaaacaat cagaataaac 33480 acaagtattt aaactctgag acagatacacaagtatttaa actccgagac agataataat 33540 tgcagttgta caacactcta tgcttctggtgtacttggca ttttgagtta cagagaatca 33600 agaaatatga ttctcacaga tgaatggttacaaatggtaa tttttttttt aatcagctca 33660 ccttatcata ggaacagata cagcaggagaagctttattt aagagacaca aacaaatata 33720 tttaccaaca agccatcaca aaaataataactaataacaa caacagtaac agctaacata 33780 cagtggttag ctatcctaag cgttttacatgcatctttag atatgcttta aaccttatag 33840 caacctgtaa ggttggtact ctttttttttctgagaggca tctcactctg tcgcccaggc 33900 tggaagtgca atggcgcgat gtcgactcactgcaacctcc acctctccag ttcaagcgat 33960 tttcctgcct cagcctcccg agtagctgggactacaggcg cccaccacca cgcctaattt 34020 ttgtattttt aatagaggca gggttttgctatgttggcca ggatggtgtc taactcctga 34080 cctcaggtga tccacctgcc tcagccttccaaagtgctga gattacaggc atgagtcacc 34140 atgcccagcc aaagtttttt gtaaggatgaaaaatatttt ttttaaaaat gaaatcaggc 34200 tgggcacagt ggctcacgcc tataatcccagcactttggg aggccaaggt tggtggatca 34260 cgaggtcagg agttcaagac cagcctgaccaacatgatga aaccccgtct ctactaaaaa 34320 tacaaaaatt agccgggcat ggtggtgtgtgcctgtaatc ccagctgctc aggaggctga 34380 ggcaggagaa tcaggaggcc ttctcaaaaaaaaaaaaaaa aaggaatcaa agcccgacat 34440 ggtggtggtg gcacatgcct gtagtcctagctatttggga gactgaggct ggaggatcac 34500 ttaaccccag gagtttgagg ctgtagaatgatactgcact tcagcctggg tgacagaggg 34560 agactccatc tcttcaaaaa aaaaatgggtgaggtggggg tggctcacgc ctgttatcca 34620 agcactttgg gaggctgagg tgggtggatcacttgagtgc aggagtttga gaccagcctg 34680 ggcaacatgg tgagacactg tctctacaaatacaaaaatt agtcaggtgt gatggtgtgt 34740 gcctataatc ccagttacta gggaggttgaggtgggagga tggatttagc ctgggaggtc 34800 gaggtgcagt gagctgtgat cccgcctctgtgctctggcc tgagtgacag agcaagactc 34860 tgtctcaaaa aaaaaaaaaa aaaaaatagaatcacatagt tggatcttgg aaatgcctgc 34920 tctgtgagta gcattcagga gtttaccacatgctagaaga tcttgggatc ttacagcccc 34980 actcatctag cccagacttt ctagtttacatttaactctt atctctcaga tgtaaatggt 35040 tctatgattc tgagattctt tggtgctccagtgcctcctg tttccctggc tggggtgtct 35100 gcaggggtgt gtaggaaggc atggatggggccaggcgcag tggctcactc acgcctgtaa 35160 tcccagcatt ttgggaggcc aaggtgggtggatcacttga gtccaggagt ttgagaccag 35220 cctggtcaac atggtgaaac cctgtctctactaaaaataa aagaaaaaat tatcagagca 35280 agtctgggcc cggtggctca cgcctgtaatcccagcactt tgggaggccg aggtggggga 35340 atcacgaggt caggagtttg agaccagcctggccaacatg gtgaaacccc atctctacta 35400 aaaatagaaa aaattagctg ggcatagtggccagcgcctg taatcccagc tactcgggag 35460 gctgaggcag gagactcact tgagccctggaggtagaggt tgcagtgagc cgagatcgtg 35520 ccactgcact ccagcccagg cgacagagtgagactccgcc tcaaaaagaa aaaaaaaaat 35580 tagctgggca tggtggtgca cgcctgtagtcccagctact tgggaggctg aggcaggaga 35640 atcacttgaa cccaggaggt aggggttgcagtgagctgag atcatgccac tgcacttcca 35700 gcctgggcta cagagcgaga ctctgtctcaaaaaaaaaaa aaaaaaagta tggatgggtt 35760 tggagggctg gctgctgagg ttgggatttggctgagtacc tatctacctt tccttactgg 35820 gcccatctgc tcccctcaga tcctggtggctttgagacac cttcacttca agaacattgt 35880 ccactgtgac ttgaaaccag aaaacgtgttgctggcatca gcagacccat ttcctcaggt 35940 cagttatgtc ccctcctgat ttggggaaatccaggcaaca ctgatggccg gggtgggggt 36000 ggggaagggg attatactaa tcaagatgtgggggcgaggc acagtggctc ttgcctgtaa 36060 tcagcatttt gagaggctga ggcaggaggatcatttgagc ccaagagttt gagaccagcc 36120 tgggcaacat agcgagacct catctatacaaaaaatgaaa aaaaaaatag ccgggaatgg 36180 tggcgtgcgc ctatagtcct agctgcttaggaggctgaga tgggaggatt gcttgagccc 36240 aggagttggt ggctgcagtg agctatgattgtgccactgc actccagcct gaataacaga 36300 gtgagagctg tctcttaaaa aaaaaaaaaaaagactgggt gcggtggctc acgcctgtaa 36360 tcccagcact ttgggaggcc gaggcgggcagttcacgagg tcaggagatc gagaccatcc 36420 tggctaacac ggtgaaaccc cttctctactaaaaatacaa aaaaaaatta gcggggcgtg 36480 gtggtgtgtg cctgtagtcc cagctacttgggaggctgag ttaggagaat ggcatgaacc 36540 cgggaggcgg agcttgcagt tagccgagatcacgccactg cactccagcc tgggtgacag 36600 agcgagagag cgagactctg tctcaaaaaaaaaaaaaaaa atatatatat atatatatat 36660 atagtttatc ccaacatata gcactttattcaacatgtag tcaacataaa aattattaag 36720 gccaggggag gtggctcatg cctataatccccgcactttg ggaggccaag atgggaagac 36780 ggcttgagac caggagttca agtctgaagtgagctatgat tgtgccactg cactccagct 36840 ggggtgacag agcaagaccc tgtctcttaaaaaagaaaca aaactcaatg aaacattctg 36900 cttgtttttc atactatgtc ttcaaaatctggtgtgtata acagttgggg aaatagattg 36960 acatgcccaa gttgttccaa acatatttaaaagttttctg gttgggcgca gcggctcatg 37020 cctataatcc cagcactttg ggaggctgaggcgggcagat cacttgaggt ctggagttgg 37080 ataccagtct ggctaacatg gcgaaaccccgtctctacta aaaatacaaa aattagctgg 37140 gcatggtggc gggaacctgt aatcccaggttctcaggagg ctgaagcagg agaattgctt 37200 gaacccagga gggtggaggt tgcggtgagccgagatcaca ccactgcact ccagcctgga 37260 cgacagacca agactcgtct caaaaaaataataataaaat aaaaatttta aaaaagatcc 37320 ataggaaagt atagatcttg gaaaagagaaagagctataa gatctgtaga aagggcagag 37380 tacctcagga aagggtggct gtcacattgagattcaggtc aggggttgag gcgtggctgg 37440 tttcaaaggt gacagaggct tcaggcttcaaggatttggg gctctatcct gcaagcaaca 37500 gtgagccaag gaagggtttt gaacagggaaaggacagtac atgaacagag ctgggaacca 37560 aggctgagag gtaggcagca gagcaagaccttgaacccag gtcttgctgg ctccaaagcc 37620 tgtccatgac cttagactgc agccattaacaatgagggta tggggccagg tgtggtgtct 37680 catgcctgta atcccagcac tttgggaggctgaggcagga ggaacacctg aggtcaggag 37740 tttgggacca gcctggctga tgtggtgaaatgtcgtctct actaaaaata caaaaattag 37800 ccaggcatgg tggcgggtcc ctgtgatcccagctattcgg gaggctgagg caggagaatt 37860 gcttgaaccc gggaggcaga ggttgcagtgagccaagatc acgctactgc actccagcct 37920 gggcgacaga gcgagactcc gtctcaaaaaaaataaaaca atgaaggaaa ggtaggcata 37980 caccatactg tctgccagct accgcagtcagcacccactc ctacctaatc cccaggaaag 38040 cctgagagga ggctgctatc aacaaccccccaatacagat gacaaaatca aggcctggag 38100 aaattaggtc cttgacctga gatcatcgagggtcattctg tgctagacac tgctcctaac 38160 acgttgcata catttctctt tcagtctaaacaagcaccct ttaaggtagg gactgttaag 38220 atctccatta tgtttcatgt tttttttgtttgttttttga gacggagtct cgctgtgtca 38280 cccaggctgg aatgcagtgg tgcgatctcggctcactgca acctctgcct cccaggttca 38340 ggcgattctc ctgcctcagc ctcctgtagtagctgggacc gcaggcgtgt gctaattttt 38400 gtatttttag tagagatggg gtttcatcgtgttggccagg ctggtctcga actcctgacc 38460 tcaaatgatc catcttcctt ggcctcccaaagtgctgaga ttgcaggcat gagccaccac 38520 gccccaatca tgtatatttt gaggctattaaaaaaaatct gcattattca aaagaggaaa 38580 cagcgaccca ttggaggtgg cagaggtatagcagcagcta gcatttattg tgcaccaact 38640 gaatgccaaa tattgtcctg tgggctttggatggtttaat tcactaacca tcatggcagt 38700 cctctgagat aggtgctctt ctgctcttcttcctatagat ggggaaactg aggcacagag 38760 gggggaagtc acctgcccag ggttgctcagctagtgagcc aaggagcctg gattcaaacc 38820 agcatccagc tttctctgga ataccatggagggtggtgtg gtggggatgc tggggtgggt 38880 gcggctccat cacctggtgg agcctccatcccttgccctc tgcaggtgaa gctgtgtgac 38940 tttggctttg ctcgcatcat cggcgagaagtcgttccgcc gctcagtggt gggcacgccg 39000 gcctacctgg cacccgaggt gctgctcaaccagggctaca accgctcgct ggacatgtgg 39060 tcagtgggcg tgatcatgta cgtcagcctcagcggcacct tccctttcaa cgaggatgag 39120 gacatcaatg accagatcca gaacgccgccttcatgtacc cggccagccc ctggagccac 39180 atctcagctg gaggtgcctg gggcccgcctaccccatggg cgggtgggtt gtggggtggg 39240 gctggagaag tgggcggagc catgagaggggggtggaccc ggaaacagcc tggcaccttg 39300 ggggtggagc ccagtgctgg ggcgggcctactggagggat gtggctacag gaggagccgt 39360 cctgtaaaag atgggctggg actcaggcctagactaggtt acttgggctg gaaaccaagt 39420 gccccagaag cgctgaggac acttggaaccttaggggggc tgagtgagac ttggcttgtc 39480 tagggtggga ccaggaaagg gactggacttgagggtacca aagggctgcg gtgaccagga 39540 gaaggggctg agcctcccaa ggcattggctgggacctgga gcctttgggt ttacgacccc 39600 aaaagggtca gccttgcaaa aaggaggcaccggtgggtag ggttgagaaa caagggcatg 39660 gctactttgc tgtgtactgg ggccgtgacttgggtgaaga tgggcctgaa gcctggggtc 39720 ggttcagtga ccaagggagc cagtctagggacgtggccgt ggagggtttc cgaagaggtc 39780 caggaacagg gctgaccctg agtcctggaagctgggagtg gatgggagtg gggaggagaa 39840 gggagccagg actgaggcag acattgcactctgcattctg gggctttggt gttgtggctg 39900 ggcctgatga agtggcaccg ggcctggtgacttgaaccta cttgggaatg ggtctgtaac 39960 tttccctgct tggaaaagtt aagtcctaaggcctggagct ttgaggctgg gtgtgggatg 40020 gcatgtttag agggccagag gcagggctaagatactgggg tgtgtcagaa gccaggagaa 40080 caagggacct gtgttggagc cagggagctcaggaagacag atggagtatg ggaagggggg 40140 ggatcattca ttcatttatt tataaccatttattcaacaa gtacattcat gtatttgtaa 40200 ccattgattc aacatgttga gtgcccacgatgtgccaggc attgactgtt ccagctctgg 40260 gaatactgtg atgacttgga cagaaggggtcaggtgcagg gtagctcatt gagtggtccg 40320 cgaagggtgg aaaggggaag ggtcctctctggagggtgcg gcttcatgga gcaggtggag 40380 cagggtgaca cggaggttgc tcggtgcaggacaagacaag gtcttggtgg tggtctaaga 40440 gcatgggccc taagcagtga gaatgtggattgacttgagt cctggagtaa tattgggggt 40500 gctcaacact ggcttttttt tttttttttgaggtggggtc tcgctctttc acccacgctg 40560 gagtgcagtg gcgtgatctc ggctcactgcaacctccacc tcttgggttc aagggattct 40620 cctgcctcag cctcccgagt aactgggattacaggcacac agcaccatgc ctggctcatg 40680 ttttatattt ttagtagaga cagggtttcgccatgttagc caggctggtc ttgaactcct 40740 gacctcaagt tatttgcccg cttcagcctcccaaagtgct gggattgcag gcataagcca 40800 tcacaccccg ccagcattgt cttttgagacccactcagaa gtccctcagt aaaagtgcat 40860 cgagtgtgca caagtgaatt taagtgtggttgcacctgtg tgaggatcac agaatcctgt 40920 gggtgttgac gggagcaggg tgcctgtgtgcaccaggcct ctcctcggat gggttcatac 40980 agtgaagcct tgtccttcat ggcttcccatcaaggagaga gcctcggatg agtgctggct 41040 tgtcttgaag cttgacattc gctagtcctctttttcacaa tgaacaggcc tatctctgag 41100 ccttctgcag gcaatggtga ctaactaccatctgatgaca ttttgttttg ttttgttttg 41160 ttttgagacg gagtttcgct tttgtcacccgggctggagt gcagtggcac gatcttggct 41220 cactgcaacc tctgcctcct gagttcaagcgattctgcct cagcctcctg agtagctggg 41280 actacaggca tgcgctacca tgcccagctaattttttgta tttttagtag agacggggtt 41340 tccgtgttgg ccaggcttgt ctcgaactcctgacctcggg tgatccaccc gcctcggcct 41400 cccaaagtgt tgggattaca ggcatgagccaccgcgccca gcctgatgac atagatgctc 41460 cctgatttgc actggggtta gataaacctgataaacccat tgcccattgt aaattgaaaa 41520 tatcataagt tggtcaggcg cagtggctgaagcccataat cccagcacct tgggaggcca 41580 aggtaggcag attgcttgag cccaggagttcaagaccagc ctgggcaatg tatctctaca 41640 aaaaatacaa aaattagccg gccatagtgacaggtgcttg tagtcccagc tggctgctca 41700 ggaggctaag gcaggagaat caattaagctggggaggtgg aggcttcagt gagcattgat 41760 cacgccactg cacttcagct tgggtaacaatgagaccctg tctcaaaaaa aaaaaaggaa 41820 gtattgtagg ttgaaaatcc atttaggccgggcgcagtgg ctcatgcctg taatcccaac 41880 aatttgggag gccaaggcag gcggattgcttgaggtcagg agttagagac cagcctggcc 41940 aatatggtga aaccccatct ctactaaaaatacaaaaagt tagcaggaca tggtgacaca 42000 cacctgtatt cctagctact tgggaggctgaggcaggaga atcacatgaa cccgggaggc 42060 ggaggttgca gtgagccaag atcgtgccattgcactccag cctgggcgac agagcgagac 42120 tctgtctcaa taaataaata agtaaaaataaaaagaatag tacaggtgta attgtatgta 42180 cctgtatatg acaaaaagaa aaaaaaaggtgacatagggg aatggggaaa ttgaagtaga 42240 gaacaggtga agagagggag ctggtgtgaacatgcatggg caggaggaga caaatttgta 42300 atgtaatgag gaaatgggtg ggtgagtgattggcacaggt gaggcttctg agccacctga 42360 gctggtgcag aaggaaggtg ttgatggcaggcaggtaggc tagggggtgc ctattggagg 42420 aggagtgacc cttgacctgt agggcttgacctgtttctct ttcctgtgca gccattgacc 42480 tcatcaacaa cctgctgcag gtgaagatgcgcaaacgcta cagcgtggac aaatctctca 42540 gccacccctg gttacaggtg atgcagggggcagggctggc ccattggctg gattggagga 42600 aggggtggga gtagatcgct tattggctaggcaggttgtg aaggatgtag gtttccttgg 42660 gtctggaatg tggctaggcc tcccattggctgggtgcagg aagagggggt ggagctaaat 42720 gtctactggc tgggtgggtt gcagagggtatggcttcacc ttcattggta cccagctctc 42780 agtggcaaac cagaggatat ccaggcactgctccaatgca gaccccaagc taaccccagt 42840 tctctcgggc ccaggagtac cagacgtggctggacctccg agagctggag gggaagatgg 42900 gagagcgata catcacgcat gagagtgacgacgcgcgctg ggagcagttt gcagcagagc 42960 atccgctgcc tgggtctggg ctgcccacggacagggatct cggtggggcc tgtccaccac 43020 aggaccacga catgcagggg ctggcggagcgcatcagtgt tctctgaggt cctgtgccct 43080 cgtccagctg ctgccctcca cagcggttcttcacaggatc ccagcaatga actgttctag 43140 ggaaagtggc ttcctgccca aactggatgggacacgtggg gagtggggtg gggggagcta 43200 tttccaaggc ccctccctgt ttccccagcaattaaaacgg actcatctct ggccccatgg 43260 ccttgatctc agcacacggc actctcgaatcattactctg ttgtaccaac atggagttca 43320 tctggaagga ggactgcctg aaaagaggaaggatggaagg ggtggggaga gaggactgat 43380 gggagaggag tcttggaagg aggacgagctggggtagaaa atatacagga agagtgccag 43440 gagagaagat gagaagggag agggaggagtaatggaggag gagttggaaa ctggggagag 43500 atggaaggaa tgtgactgga gggtagagaacttggagaaa aagtaatctc atggtttgtg 43560 atgactgatt ttttatttgg tggtggtgttactactaatc acaactatta attcaggctg 43620 ggtgtggtgg ctcatgccta taatcccagcaatttgggag gccgaggcag gcagatccct 43680 tagatctcag gagtttgaga gcagcctggccaacgtggtg aaactccctt tctacaaaaa 43740 gttcaaaaat tagccaagtg tggtggcttgcacctgtggt cccagctact tggaggttga 43800 ggctagagga tcgcttgagc ccaggaagcagagattgcag tgagccaaga tcacacacca 43860 ctgcactcta gcctgggcaa gagagtgagaccctgtctca aaagtcaaat aataaaatgc 43920 agttagccca agtctgatcc atactagaaa43950 4 894 PRT Homo sapiens 4 Ala Ala Ala Ala Ala Ala Ala Ala Leu ValPro Gly Ser Gly Pro Gly 1 5 10 15 Pro Ala Pro Phe Leu Ala Pro Val AlaAla Pro Val Gly Gly Ile Ser 20 25 30 Phe His Leu Gln Ile Gly Leu Ser ArgGlu Pro Val Leu Leu Leu Gln 35 40 45 Asp Ser Ser Gly Asp Tyr Ser Leu AlaHis Val Arg Glu Met Ala Cys 50 55 60 Ser Ile Val Asp Gln Lys Phe Pro GluCys Gly Phe Tyr Gly Met Tyr 65 70 75 80 Asp Lys Ile Leu Leu Phe Arg HisAsp Pro Thr Ser Glu Asn Ile Leu 85 90 95 Gln Leu Val Lys Ala Ala Ser AspIle Gln Glu Gly Asp Leu Ile Glu 100 105 110 Val Val Leu Ser Arg Ser AlaThr Phe Glu Asp Phe Gln Ile Arg Pro 115 120 125 His Ala Leu Phe Val HisSer Tyr Arg Ala Pro Ala Phe Cys Asp His 130 135 140 Cys Gly Glu Met LeuTrp Gly Leu Val Arg Gln Gly Leu Lys Cys Glu 145 150 155 160 Gly Cys GlyLeu Asn Tyr His Lys Arg Cys Ala Phe Lys Ile Pro Asn 165 170 175 Asn CysSer Gly Val Arg Arg Arg Arg Leu Ser Asn Val Ser Leu Thr 180 185 190 GlyVal Ser Thr Ile Arg Thr Ser Ser Ala Glu Leu Ser Thr Ser Ala 195 200 205Pro Asp Glu Pro Leu Leu Gln Lys Ser Pro Ser Glu Ser Phe Ile Gly 210 215220 Arg Glu Lys Arg Ser Asn Ser Gln Ser Tyr Ile Gly Arg Pro Ile His 225230 235 240 Leu Asp Lys Ile Leu Met Ser Lys Val Lys Val Pro His Thr PheVal 245 250 255 Ile His Ser Tyr Thr Arg Pro Thr Val Cys Gln Tyr Cys LysLys Leu 260 265 270 Leu Lys Gly Leu Phe Arg Gln Gly Leu Gln Cys Lys AspCys Arg Phe 275 280 285 Asn Cys His Lys Arg Cys Ala Pro Lys Val Pro AsnAsn Cys Leu Gly 290 295 300 Glu Val Thr Ile Asn Gly Asp Leu Leu Ser ProGly Ala Glu Ser Asp 305 310 315 320 Val Val Met Glu Glu Gly Ser Asp AspAsn Asp Ser Glu Arg Asn Ser 325 330 335 Gly Leu Met Asp Asp Met Glu GluAla Met Val Gln Asp Ala Glu Met 340 345 350 Ala Met Ala Glu Cys Gln AsnAsp Ser Gly Glu Met Gln Asp Pro Asp 355 360 365 Pro Asp His Glu Asp AlaAsn Arg Thr Ile Ser Pro Ser Thr Ser Asn 370 375 380 Asn Ile Pro Leu MetArg Val Val Gln Ser Val Lys His Thr Lys Arg 385 390 395 400 Lys Ser SerThr Val Met Lys Glu Gly Trp Met Val His Tyr Thr Ser 405 410 415 Lys AspThr Leu Arg Lys Arg His Tyr Trp Arg Leu Asp Ser Lys Cys 420 425 430 IleThr Leu Phe Gln Asn Asp Thr Gly Ser Arg Tyr Tyr Lys Glu Ile 435 440 445Pro Leu Ser Glu Ile Leu Ser Leu Glu Pro Val Lys Thr Ser Ala Leu 450 455460 Ile Pro Asn Gly Ala Asn Pro His Cys Phe Glu Ile Thr Thr Ala Asn 465470 475 480 Val Val Tyr Tyr Val Gly Glu Asn Val Val Asn Pro Ser Ser ProSer 485 490 495 Pro Asn Asn Ser Val Leu Thr Ser Gly Val Gly Ala Asp ValAla Arg 500 505 510 Met Trp Glu Ile Ala Ile Gln His Ala Leu Met Pro ValIle Pro Lys 515 520 525 Gly Ser Ser Val Gly Thr Gly Thr Asn Leu His ArgAsp Ile Ser Val 530 535 540 Ser Ile Ser Val Ser Asn Cys Gln Ile Gln GluAsn Val Asp Ile Ser 545 550 555 560 Thr Val Tyr Gln Ile Phe Pro Asp GluVal Leu Gly Ser Gly Gln Phe 565 570 575 Gly Ile Val Tyr Gly Gly Lys HisArg Lys Thr Gly Arg Asp Val Ala 580 585 590 Ile Lys Ile Ile Asp Lys LeuArg Phe Pro Thr Lys Gln Glu Ser Gln 595 600 605 Leu Arg Asn Glu Val AlaIle Leu Gln Asn Leu His His Pro Gly Val 610 615 620 Val Asn Leu Glu CysMet Phe Glu Thr Pro Glu Arg Val Phe Val Val 625 630 635 640 Met Glu LysLeu His Gly Asp Met Leu Glu Met Ile Leu Ser Ser Glu 645 650 655 Lys GlyArg Leu Pro Glu His Ile Thr Lys Phe Leu Ile Thr Gln Ile 660 665 670 LeuVal Ala Leu Arg His Leu His Phe Lys Asn Ile Val His Cys Asp 675 680 685Leu Lys Pro Glu Asn Val Leu Leu Ala Ser Ala Asp Pro Phe Pro Gln 690 695700 Val Lys Leu Cys Asp Phe Gly Phe Ala Arg Ile Ile Gly Glu Lys Ser 705710 715 720 Phe Arg Arg Ser Val Val Gly Thr Pro Ala Tyr Leu Ala Pro GluVal 725 730 735 Leu Arg Asn Lys Gly Tyr Asn Arg Ser Leu Asp Met Trp SerVal Gly 740 745 750 Val Ile Ile Tyr Val Ser Leu Ser Gly Thr Phe Pro PheAsn Glu Asp 755 760 765 Glu Asp Ile His Asp Gln Ile Gln Asn Ala Ala PheMet Tyr Pro Pro 770 775 780 Asn Pro Trp Lys Glu Ile Ser His Glu Ala IleAsp Leu Ile Asn Asn 785 790 795 800 Leu Leu Gln Val Lys Met Arg Lys ArgTyr Ser Val Asp Lys Thr Leu 805 810 815 Ser His Pro Trp Leu Gln Asp TyrGln Thr Trp Leu Asp Leu Arg Glu 820 825 830 Leu Glu Cys Lys Ile Gly GluArg Tyr Ile Thr His Glu Ser Asp Asp 835 840 845 Leu Arg Trp Glu Lys TyrAla Gly Glu Gln Arg Leu Gln Tyr Pro Thr 850 855 860 His Leu Ile Asn ProSer Ala Ser His Ser Asp Thr Pro Glu Thr Glu 865 870 875 880 Glu Thr GluMet Lys Ala Leu Gly Glu Arg Val Ser Ile Leu 885 890

That which id claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b)) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:l or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids.
 3. An isolated antibody that selectivelybinds to a peptide of claim
 2. 4. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes an amino acid sequence shownin SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO:2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO:2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO:2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO:2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human kinase protein, said methodcomprising administering to a patient a pharmaceutically effectiveamount of an agent identified by the method of claim
 16. 19. A methodfor identifying a modulator of the expression of a peptide of claim 2,said method comprising contacting a cell expressing said peptide with anagent, and determining if said agent has modulated the expression ofsaid peptide.
 20. An isolated human kinase peptide having an amino acidsequence that shares at least 70% homology with an amino acid sequenceshown in SEQ ID NO:2.
 21. A peptide according to claim 20 that shares atleast 90 percent homology with an amino acid sequence shown in SEQ IDNO:2.
 22. An isolated nucleic acid molecule encoding a human kinasepeptide, said nucleic acid molecule sharing at least 80 percent homologywith a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 23. A nucleicacid molecule according to claim 22 that shares at least 90 percenthomology with a nucleic acid molecule shown in SEQ ID NOS: 1 or 3.